Methods for the diagnosis of fetal disease

ABSTRACT

Methods are provided for detecting an aneuploidy in a fetus. These methods can be used to detect trisomy 13, 8 or 21, amongst other aneupoloidies. In some embodiments, the methods include selectively purifying fetal DNA from a maternal biological sample using the methylation status of a CpG containing genomic sequence and genotyping the fetus using the purified fetal DNA, thereby detecting aneuploidy in the fetus.

CROSS REFERENCE TO RELATED APPLICATIONS

This claims the benefit of U.S. Provisional Application No. 61/361,824,filed Jul. 6, 2010, which is incorporated by reference herein in itsentirety.

FIELD

This related to the field of prenatal diagnosis, specifically to thediagnosis of fetal aneuploidy, such as an aneuploidy of chromosome 13,18, 21, X or Y.

BACKGROUND

Definitive prenatal diagnosis of fetal aneuploidy is currently performedusing chorionic villus sampling (CVS) or amniocentesis. These areinvasive, expensive and somewhat risky approaches that must be performedby experienced clinicians and are generally only offered to expectantmothers considered to be at elevated risk of carrying a geneticallyabnormal fetus. Low cost minimally invasive screening is routinely usedacross the maternal age range but this is based upon the quantificationof serum proteins that are surrogate markers of the underlying geneticabnormality and do not achieve desirable levels of sensitivity andspecificity (Wapner et al., N. Eng J. Med. 349: 1405-13, 2003; Alfirevicand Neilson, BMJ 326: 811-2, 2004; Malone et al., N Engl. J. Med 353:2001-11, 2005).

An attractive alternative involves the analysis of placentally-derivednucleic acids in maternal plasma to characterize specific features ofthe fetal genome (Lo, Ann. NY. Acad. Sci. 1137: 140-3, 2008). However,because of the technical challenge of distinguishing maternallyinherited fetal alleles from endogenous maternal DNA this approach haslargely been limited to the detection of paternally inheriteddisease-causing mutations (Zimmermann et al., Methods Mol Med 132:43-49, 2007; Li et al., Prenat Diag 27: 11-7, 2007). Differential DNAmethylation patterns between the placental and maternal leukocytegenomes at distinct loci have been used to determine the fetal genotype(Lo et al., PNAS 104: 13116-21, 2007; Lo et al., Nat Med 13:218-23,2007). Specific polymerase chain reaction (PCR)-based assays were usedthat amplify a given methylated (placental), but not an unmethylated(maternal) locus (or visa versa) in DNA obtained from maternal plasma(Tsui et al., Prenat Diag 27: 1212-8, 2007; Tong et al., Clin Chem 53:1906-14, 2007; Chan et al., Clin Chem 52: 2211-8, 2006; Tong et al.,Clin Chem 52: 2194-202, 2006). However, a need remains for sensitive andspecific non-invasive methods that can be used to diagnose fetalconditions, such as aneuploidy, using the placental methylome.

SUMMARY

Methods are provided for detecting a chromosome abnormality such as ananeuploidy in a fetus. These methods can be used to detect trisomy 13,8, 21, X and/or Y, amongst other aneupoloidies. In some embodiments, themethods detect a trisomy of chromosome 13, 8, and/or 21. In otherembodiments the methods detect Turner's Syndrome (XO).

In some embodiments, the methods include selectively purifying fetal DNAfrom a maternal biological sample using the methylation status of a CpGcontaining genomic sequence. The allele frequency is then determined inthe purified fetal DNA, thereby detecting aneuploidy in the fetus.

In some embodiments, methods are provided for detecting an aneuploidy ina fetus. The methods include (a) selectively purifying fetal DNA from amaternal biological sample using the methylation status of a CpGcontaining genomic sequence, wherein the CpG-containing genomic sequenceis at least 15 nucleotides in length, comprises at least one CpGdinucleotide, and is within a region on chromosome 13, 18 or 21, andwherein the CpG-containing genomic sequence comprises at least 15nucleotides of at least one of the nucleic acid sequences set forth asany one of SEQ ID NOs: 1-68 or 83-85; and (b) genotyping the fetus usingthe purified fetal DNA, thereby detecting aneuploidy in the fetus.

In some, but not all, embodiments, the fetus is genotyped using agenetic marker, such as a single nucleotide polymorphism (SNP) that isgenetically linked to any one of SEQ ID NOs: 1-68 or 83-85. In someexamples, the SNP is within about 150 base pairs of at least one of SEQID NOs: 1-68 or 83-85. Other genetic markers are also of use, such as ashort tandem repeat (STR).

In some embodiments, the allelic ratio is determined. In some examples,an allelic ratio of 1:2 or 2:1 indicates that the fetus is aneuploid. Inother examples, an allelic ratio of 0:1 or 1:0 indicates that the fetusis aneuploidy.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the reaction scheme used to preparelibraries of genomic DNA fragments that are depleted for hypomethylatedCpG sites in either CVS or MBC tissues.

FIG. 2A-B is a set of graphs. FIG. 2A is a summary of pyrosequencinganalysis for four CpG sites across chromosomes 13, 18 and 21. Samplen=10 CVS and 10 MBC. FIG. 2 b is a summary of Mass Array/Epityperanalysis for CpG sites across chromosomes 13, 18 and 21. Sample n=23 CVSand 23 MBC. For both data sets, fully methylated control samples wereobtained from Millipore (part number S7821). The Y axis is methylationrate, i.e., the proportion of CpG sites that are methylated. Thewhiskers above each bar are the upper boundaries of the confidenceintervals of the estimated methylation rates.

FIG. 3 is a presentation of data from a real time PCR analysis of twodifferent maternal plasma samples. Each cluster of four dots represents4 replicates for each condition. The cluster represented by the * isfrom maternal plasma DNA that was not cut with HpaII. Therefore there isno selection for fetal DNA and the overwhelming signal read by theinstrument is the TT genotype. When this DNA is cut with HpaII the fetalDNA is preserved and the maternal DNA lost because it will not amplify(@). The bottom two clusters are from a second plasma sample. The lowestcluster is the uncut (sham) sample and clearly shows the maternal CCgenotype (#). The higher of the two is the fetal genotype (CT) that hasis detectable only after selection of fetal DNA via HpaII digestion ($).The nucleic acid sequence reference is SEQ ID NO: 1.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile [8123-83157-03sequence.txt, 64 kB, Jul. 5, 2011], and incorporatedby reference herein. In the accompanying sequence listing:

SEQ ID NOs: 1-68 and 83 are nucleic acid sequences of regions of humanchromosomal DNA that are methylated in a fetal genome as compared to amaternal genome.

SEQ ID NOs: 69-82 are primer sequences.

DETAILED DESCRIPTION

Methods are provided herein for detecting an aneuploidy in a fetus.These methods include selectively purifying fetal DNA from a maternalbiological sample using the methylation status of a CpG containinggenomic sequence. The copy number of a DNA sequence included in thefetal genome is then determined.

In some embodiments, the CpG-containing genomic sequence is at least 15nucleotides in length. In some embodiments, the CpG containing sequencecomprises at least one CpG dinucleotide, and is within a region onchromosome 13, 18, 21, X or Y. In specific examples, the CpG-containinggenomic sequence comprises at least 15 nucleotides of at least one ofthe nucleic acid sequences set forth as SEQ ID NOs: 1-68 or 83-85. Inother specific non-limiting examples, the methylation status of theentirety of one or more of SEQ ID NO:s: 1-68 or 83-85 can also bedetermined.

These methods can include genotyping the fetus using the purified fetalDNA, thereby detecting aneuploidy of chromosome 13, 18 or 21 in thefetus. However, these methods can be applied to other chromosomes,including the X chromosome.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Allelic ratio: An allele is one DNA sequence of a given genetic locus.The number of each alleles in an individual is an allelic ratio.Generally, an allelic ratio of 2:0 or 0:2 is a homozygote, and anallelic ratio of 1:1 is a heterozygote at the locus. An allelic ratio of1:2 or 2:1 indicates that a subject, such as a fetus, is aneuploid. Anallelic ratio of 1:0 or 0:1, such as for the X chromosome ratio in asubject that is phenotypically female, can also indicate that thesubject is aneuploid.

Amplification: To increase the number of copies of a nucleic acidmolecule. The resulting amplification products are called “amplicons.”Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification is thepolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Other examples of in vitro amplification techniques include quantitativereal-time PCR; reverse transcriptase PCR (RT-PCR); real-time PCR (rtPCR); real-time reverse transcriptase PCR (rt RT-PCR); nested PCR;strand displacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see PCT Publication No.WO 90/01069); ligase chain reaction amplification (see European patentpublication No. EP-A-320 308); gap filling ligase chain reactionamplification (see U.S. Pat. No. 5,427,930); coupled ligase detectionand PCR (see U.S. Pat. No. 6,027,889); and NASBA™ RNA transcription-freeamplification (see U.S. Pat. No. 6,025,134), amongst others.

Allele (Haplotype): A 5′ to 3′ sequence of nucleotides found at a set ofone or more polymorphic sites in a locus on a single chromosome from asingle individual. “Allelic pair” is the two alleles found for a locusin a single individual. With regard to a population, alleles are theordered, linear combination of polymorphisms (e.g., single nucleotidepolymorphisms (SNPs) in the sequence of each faun of a gene (onindividual chromosomes) that exist in the population. “Haplotyping” is aprocess for determining one or more alleles in an individual andincludes use of family pedigrees, molecular techniques and/orstatistical inference. “Haplotype data” or “allele data” is theinformation concerning one or more of the following for a specific gene:a listing of the allelic pairs in an individual or in each individual ina population; a listing of the different alleles in a population;frequency of each allele in that or other populations, and any knownassociations between one or more alleles and a trait.

Aneuploidy: An abnormal number of chromosomes. Monosomy refers to thepresence of only one chromosome, wherein two copies is normal. Monosomyof the X chromosome (45,X) causes Turner's syndrome. Trisomy refers tothe presence of three copies (instead of the normal two) of specificchromosomes. Trisomy 21 causes Down's syndrome. Tripsome 10 and Trisomy31, known as Edwards and Patau Syndrome, respectively, are two autosomalabnormalities. Trisomy X has also been observed in humans (47, XXX).

Germline aneuploidy can be detected through karyotyping, a process inwhich a sample of cells is fixed and stained to create the typical lightand dark chromosomal banding pattern and a picture of the chromosomes isanalyzed. Other techniques include Fluorescence In Situ Hybridization(FISH), Quantitative Polymerase Chain Reaction (PCR) of Short TandemRepeats, Quantitative Fluorescence PCR (QF-PCR), Quantitative Real-timePCR(RT-PCR) dosage analysis, Quantitative Mass Spectrometry of SingleNucleotide Polymorphisms, and Comparative Genomic Hybridization (CGH).

Array: An arrangement of molecules, such as biological macromolecules(such as polypeptides or nucleic acids) or biological samples (such astissue sections), in addressable locations on or in a substrate. A“microarray” is an array that is miniaturized so as to require or beaided by microscopic examination for evaluation or analysis. Arrays aresometimes called DNA chips or biochips.

The array of molecules (“features”) makes it possible to carry out avery large number of analyses on a sample at one time. In certainexample arrays, one or more molecules (such as an oligonucleotide probe)will occur on the array a plurality of times (such as twice), forinstance to provide internal controls. The number of addressablelocations on the array can vary, for example from a few (such as three)to at least six, at least 20, at least 25, or more. In particularexamples, an array includes nucleic acid molecules, such asoligonucleotide sequences that are at least 15 nucleotides in length,such as about 15-40 nucleotides in length, such as at least 18nucleotides in length, at least 21 nucleotides in length, or even atleast 25 nucleotides in length. In one example, the molecule includesoligonucleotides attached to the array via their 5′- or 3′-end.

Within an array, each arrayed sample is addressable, in that itslocation can be reliably and consistently determined within at least twodimensions of the array. The feature application location on an arraycan assume different shapes. For example, the array can be regular (suchas arranged in uniform rows and columns) or irregular. Thus, in orderedarrays the location of each sample is assigned to the sample at the timewhen it is applied to the array, and a key may be provided in order tocorrelate each location with the appropriate target or feature position.Often, ordered arrays are arranged in a symmetrical grid pattern, butsamples could be arranged in other patterns (such as in radiallydistributed lines, spiral lines, or ordered clusters). Addressablearrays usually are computer readable, in that a computer can beprogrammed to correlate a particular address on the array withinformation about the sample at that position (such as hybridization orbinding data, including for instance signal intensity). In some examplesof computer readable formats, the individual features in the array arearranged regularly, for instance in a Cartesian grid pattern, which canbe correlated to address information by a computer.

Bi-Allelic Single Nucleotide Polymorphism: A polymorphism (onenucleotide) that differs between the two alleles in an individual. Thus,the individual is heterozygous at this genetic loci.

Bisulfite: All types of bisulfites, such as sodium bisulfate, that arecapable of chemically converting a cytosine (C) to a uracil (U) withoutchemically modifying a methylated cytosine and therefore can be used todifferentially modify a DNA sequence based on the methylation status ofthe DNA.

Chromosomal abnormality: A chromosome with DNA deletions or duplicationsand chromosomal aneuploidy. The term also encompasses translocation ofextra chromosomal sequences to other chromosomes.

Chromosomal aneuploidy or aneuploidy: The abnormal presence(hyperploidy) or absence (hypoploidy) of a chromosome, such aschromosome 13, 18 or 21. In some cases, the abnormality can involve morethan one chromosome, or more than one portion of one or morechromosomes. The most common chromosome aneuploidy is trisomy, such astrisomy 21, where the genome of an afflicted patient has threechromosomes 21, as compared to two chromosomes 21. In rarer cases, thepatient may have an extra piece of chromosome 21 (less than full length)in addition to the normal pair. In yet other cases, a portion ofchromosome 21 may be translocated to another chromosome, such aschromosome 14. In this example, chromosome 21 is referred as the“chromosome relevant to the chromosomal aneuploidy” and a second,chromosome that is present in the normal pair in the patient's genome,for example chromosome 1, is a “reference chromosome.” There are alsocases where the number of a relevant chromosome is less than the normalnumber of 2. Turner syndrome is one example of a chromosomal aneuploidywhere the number of X chromosome in a female subject has been reducedfrom two to one.

CpG-containing genomic sequence: A segment of DNA sequence at a definedlocation in the genome of an individual such as a human fetus or apregnant woman. Typically, a “CpG-containing genomic sequence” is atleast 15 nucleotides in length and contains at least one cytosine. Insome embodiments, a CpG containing sequence can be at least 30, 50, 80,100, 150, 200, 250, or 300 nucleotides in length and contains at least2, 5, 10, 15, 20, 25, or 30 cytosines. For any specific “CpG-containinggenomic sequence” at a given location, for example, within a regioncentering around a given genetic locus on chromosome 21 nucleotidesequence variations can exist from individual to individual and fromallele to allele even for the same individual. Typically, such a regioncentering around a defined genetic locus (e.g., a CpG island) containsthe locus as well as upstream and/or downstream sequences. Each of theupstream or downstream sequence (counting from the 5′ or 3′ boundary ofthe genetic locus, respectively) can be as long as 1 kb, in other casesmay be as long as 5 kb, 2 kb, 750 bp, 500 bp, 200 bp, or 100 bp. A“CpG-containing genomic sequence” can encompass a coding or anon-coding, nucleic acid sequence, and thus can include a nucleotidesequence transcribed (or not transcribed) for protein production. Thus,a CpG containing genomic sequence can be a nucleotide sequence can be aprotein-coding sequence, a non protein-coding sequence or a combinationthereof.

CpG island: A segment of DNA sequence in which the frequency of CpGdinucleotide sequences is higher than other dinucleotide sequences.Generally, a CpG island is found in a genome that has a minimal length,a minimal GC content, and a minimal ratio of observed CpGfrequency/expected CpG frequency (OCF/ECF).

In one embodiment, a CpG island has (1) at least 200 nucleotides inlength, (2) has a greater than 50% GC content, and (3) an OCF/ECF ratiogreater than 0.6 (see Takai et al., Proc. Natl. Acad. Sci. U.S.A.99:3740-3745, 2002). In another embodiment, a CpG island has (1) atleast 400 nucleotides in length; (2) a greater than 50% GC content; and(3) an OCF/ECF ratio greater than 0.6 (see Yamada et al. (GenomeResearch 14:247-266, 2004). A “CpG island” on chromosome 13, 18 or 21can fits the CpG island profiles provided by any one of the currentlyavailable computational programs designed for scanning chromosomes basedon the above stated criteria, encompassing results obtained when usingwindow sizes of 100, 200, or 300 nucleotides and shift or step sizes of1, 2, or 3 nucleotides in the screening process. The individual CpGislands named in this disclosure are further defined by including thesequence, but can also be identified by contig number, version andregion at GENBANK®, chromosomal location relative to the chromosome 13,18 or 21 sequence, respectively, of the Human May 2004 (hg17) assemblyof the UCSC Genome Browser (See the UCSC website).

Control DNA: Genomic DNA obtained from an individual that is used forcomparative purposes, such as DNA from a healthy individual who does nothave a chromosomal abnormality. In some embodiments, a control DNAsample can be obtained from plasma of a female carrying a healthy fetuswho does not have a chromosomal abnormality, which can serve as anegative control. When certain chromosome anomalies are known, thecontrol can also be established standards that are indicative of aspecific disease or condition.

To screen for three different chromosomal aneuploidies in a maternalplasma of a pregnant female, a panel of control DNAs that have beenisolated from plasma of mothers who are known to carry a fetus with, forexample, chromosome 13, 18, or 21 trisomy, and a mother who is pregnantwith a fetus who does not have a chromosomal abnormality can be used asa positive control.

Copy number: The number of copies of a section of DNA in a genome. Copynumber analysis usually refers to the process of analyzing data producedby a test for DNA copy number variation in patient's sample. Suchanalysis helps detect chromosomal copy number variation that may causeor may increase risks of various critical disorders. Copy numbervariation can be detected with various types of tests, including, butnot limited to, such as methylation status, fluorescent in situhybridization, comparative genomic hybridization high-resolutionarray-based tests based on array comparative genomic and SNP arraytechnologies. The methods disclosed herein can be used to determine thecopy number of a specific locus of interest.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal (termination codon). The term codon is also used forthe corresponding (and complementary) sequences of three nucleotides inthe mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule. Thus, a reference to the nucleic acidmolecule that encodes a protein, or a fragment thereof, encompasses boththe sense strand and its reverse complement. Thus, for instance, it isappropriate to generate probes or primers from the reverse complementsequence of the disclosed nucleic acid molecules.

Differentially Modifies (methylated or non-methylated DNA): A reagentthat modifies methylated or non-methylated DNA, respectively, in aprocess through which distinguishable products result from methylatedand non-methylated DNA, thereby allowing the identification of the DNAmethylation status. Such processes may include, but are not limited to,chemical reactions (such as conversion by bisulfate) and enzymatictreatment (such as cleavage by a methylation-dependent endonuclease), oran antibody that specifically binds a methylated (or non-methylated) DNAsequence. Thus, an enzyme that preferentially cleaves or digestsmethylated DNA is one capable of cleaving or digesting a DNA molecule ata significantly higher efficiency when the DNA is methylated, whereas anenzyme that preferentially cleaves or digests unmethylated DNA exhibitsa significantly higher efficiency when the DNA is not methylated.

Epigenetic status: Any structural feature at the molecular level of anucleic acid (e.g., DNA or RNA) other than the primary nucleotidesequence. For instance, the epigenetic state of a genomic DNA mayinclude its secondary or tertiary structure determined or influenced by,for example, its methylation pattern or its association with cellularproteins, such as histones, and the modifications of such proteins, suchas acetylation, deacetylation, and methylation.

Gene: A segment of DNA that contains the coding sequence for a protein,wherein the segment may include promoters, exons, introns, and otheruntranslated regions that control expression.

Genotype: An unphased 5′ to 3′ sequence of nucleotide pair(s) found at aset of one or more polymorphic sites in a locus on a pair of homologouschromosomes in an individual. “Genotyping” is a process for determininga genotype of an individual.

Genomic target sequence: A sequence of nucleotides located in aparticular region in the human genome that corresponds to one or morespecific genetic abnormalities, such as a nucleotide polymorphism, adeletion, an insertion, or an amplification. The target can be forinstance a coding sequence; it can also be the non-coding strand thatcorresponds to a coding sequence. The target can also be a non-codingsequence, such as an intronic sequence.

Heterozygous: An organism is heterozygous for a particular allele whentwo different alleles occupy the gene's position (locus) on thehomologous chromosomes. The cell or organism is called a heterozygote.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidsconsist of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between two distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence. For example, anoligonucleotide can be complementary to a specific genetic locus, so itspecifically hybridizes with a mutant allele (and not the referenceallele) or so that it specifically hybridizes with a reference allele(and not the mutant allele).

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target, such that the target can be distinguished.The oligonucleotide or oligonucleotide analog need not be 100%complementary to its target sequence to be specifically hybridizable. Anoligonucleotide or analog is specifically hybridizable when binding ofthe oligonucleotide or analog to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA, and thereis a sufficient degree of complementarity to avoid non-specific bindingof the oligonucleotide or analog to non-target sequences underconditions where specific binding is desired, for example underphysiological conditions in the case of in vivo assays or systems. Suchbinding is referred to as specific hybridization. In one example, anoligonucleotide is specifically hybridizable to DNA or RNA nucleic acidsequences including an allele of a gene, wherein it will not hybridizeto nucleic acid sequences containing a polymorphism.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though wash times Alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11.

The following is an exemplary set of hybridization conditions and is notlimiting:

Very High Stringency (Detects Sequences that Share at Least 90%Identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share at Least 80% Identity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share at Least 50% Identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Increase or a Decrease: A significantly significant positive or negativechange, respectively, in quantity from a control value. An increase is apositive change, such as a 50%, 100%, 200%, 300%, 400% or 500% increaseas compared to the control value. A decrease is a negative change, suchas a 50%, 100%, 200%, 300%, 400% or 500% decrease as compared to acontrol value.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Locus: A location on a chromosome or DNA molecule corresponding to agene or a physical or phenotypic feature, where physical featuresinclude polymorphic sites.

Maternal allele frequency: The ratio, represented as a percent, of amaternal allele to the total amount of alleles present (both paternaland maternal). The term “paternal allele frequency” refers to the ratio,represented as a percent, of a paternal allele to the total amount ofalleles present (both paternal and maternal).

Methylation status: The state of methylation of a genomic sequence. Thisrefers to the characteristics of a DNA segment at a particular genomiclocus relevant to methylation. Such characteristics include, but are notlimited to, whether any of the cytosine (C) residues within this DNAsequence are methylated, location of methylated C residue(s), percentageof methylated C at any particular stretch of residues, and allelicdifferences in methylation. The methylation profile affects the relativeor absolute concentration of methylated C or unmethylated C at anyparticular stretch of residues in a biological sample.

Methyl-sensitive enzymes: DNA restriction endonucleases that aredependent on the methylation state of their DNA recognition site foractivity. For example, there are methyl-sensitive enzymes that cleave attheir DNA recognition sequence only if it is not methylated. Thus, anunmethylated DNA sample will be cut into smaller fragments than amethylated DNA sample. Similarly, a hypermethylated DNA sample will notbe cleaved. In contrast, there are methyl-sensitive enzymes that cleaveat their DNA recognition sequence only if it is methylated. As usedherein, the terms “cleave”, “cut” and “digest” are used interchangeably.

Methyl-sensitive enzymes that digest unmethylated DNA suitable for usein methods of the invention include, but are not limited to, HpaII,HhaI, MaeII, BstUI and AciI. One enzyme is HpaII that cuts only theunmethylated sequence CCGG. Enzymes that digest only methylated DNAinclude, but are not limited to, DpnI, which cuts at a recognitionsequence GATC, and McrBC, which belongs to the family of AAA proteins(New England BioLabs, Inc., Beverly, Mass.).

Cleavage methods and procedures for selected restriction enzymes forcutting DNA at specific sites are well known to the skilled artisan. Forexample, many suppliers of restriction enzymes provide information onconditions and types of DNA sequences cut by specific restrictionenzymes, including New England BioLabs, Promega Corporation,Boehringer-Mannheim, and the like. Sambrook et al. (See Sambrook et al.,Molecular Biology: A Laboratory Approach, Cold Spring Harbor, N.Y. 1989)provide a general description of methods for using restriction enzymesand other enzymes.

Mutation: Any change of a nucleic acid sequence as a source of geneticvariation. For example, mutations can occur within a gene or chromosome,including specific changes in non-coding regions of a chromosome, forinstance changes in or near regulatory regions of genes. Types ofmutations include, but are not limited to, base substitution pointmutations (which are either transitions or transversions), deletions,and insertions. Missense mutations are those that introduce a differentamino acid into the sequence of the encoded protein; nonsense mutationsare those that introduce a new stop codon; and silent mutations arethose that introduce the same amino acid often with a base change in thethird position of the codon. In the case of insertions or deletions,mutations can be in-frame (not changing the frame of the overallsequence) or frame shift mutations, which may result in the misreadingof a large number of codons (and often leads to abnormal termination ofthe encoded product due to the presence of a stop codon in thealternative frame).

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

In several examples, oligonucleotides and oligonucleotide analogs caninclude linear sequences up to about 200 nucleotides in length, forexample a sequence (such as DNA or RNA) that is at least 6 bases, forexample at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200bases long, or from about 6 to about 70 bases, for example about 10-25bases, such as 12, 15 or 20 bases.

Polymorphic marker: A segment of genomic DNA that exhibits heritablevariation in a DNA sequence between individuals. Such markers include,but are not limited to, single nucleotide polymorphisms (SNPs),restriction fragment length polymorphisms (RFLPs), short tandem repeats,such as di-, tri- or tetra-nucleotide repeats (STRs), and the like.Polymorphic markers can be used to specifically differentiate between amaternal and paternal allele in the enriched fetal nucleic acid sample.

A “methyl-polymorphic marker” refers to a polymorphic marker that isadjacent to differentially methylated DNA regions of fetal and maternalDNA. The term adjacent refers to a marker that is within 1-3000 basepairs, preferably 1000 base pairs, such as 150 base pairs, 100 basepairs or 50 base pairs from a differentially methylated nucleotide.

Polymorphism: A variation in a gene sequence. The polymorphisms can bethose variations (DNA sequence differences) which are generally foundbetween individuals or different ethnic groups and geographic locationswhich, while having a different sequence, produce functionallyequivalent gene products. Typically, the term can also refer to variantsin the sequence which can lead to gene products that are notfunctionally equivalent. Polymorphisms also encompass variations whichcan be classified as alleles and/or mutations which can produce geneproducts which may have an altered function. Polymorphisms alsoencompass variations which can be classified as alleles and/or mutationswhich either produce no gene product or an inactive gene product or anactive gene product produced at an abnormal rate or in an inappropriatetissue or in response to an inappropriate stimulus. Alleles are thealternate forms that occur at the polymorphism.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule or protein that islinked to the variation.

Probes and primers: A probe comprises an isolated nucleic acid capableof hybridizing to a target nucleic acid. A detectable label or reportermolecule can be attached to a probe or primer. Typical labels includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, for example in Sambrook et al. (InMolecular Cloning: A Laboratory Manual, CSHL, New York, 1989) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998).

In a particular example, a probe includes at least one fluorophore, suchas an acceptor fluorophore or donor fluorophore. For example, afluorophore can be attached at the 5′- or 3′-end of the probe. Inspecific examples, the fluorophore is attached to the base at the 5′-endof the probe, the base at its 3′-end, the phosphate group at its 5′-endor a modified base, such as a T internal to the probe.

Probes are generally at least 15 nucleotides in length, such as at least15, at least 16, at least 17, at least 18, at least 19, least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29, at least 30, at least 31, atleast 32, at least 33, at least 34, at least 35, at least 36, at least37, at least 38, at least 39, at least 40, at least 41, at least 42, atleast 43, at least 44, at least 45, at least 46, at least 47, at least48, at least 49, at least 50 at least 51, at least 52, at least 53, atleast 54, at least 55, at least 56, at least 57, at least 58, at least59, at least 60, at least 61, at least 62, at least 63, at least 64, atleast 65, at least 66, at least 67, at least 68, at least 69, at least70, or more contiguous nucleotides complementary to the target nucleicacid molecule, such as 20-70 nucleotides, 20-60 nucleotides, 20-50nucleotides, 20-40 nucleotides, or 20-30 nucleotides.

Primers are short nucleic acid molecules, for instance DNAoligonucleotides are 10 nucleotides or more in length, which can beannealed to a complementary target nucleic acid molecule by nucleic acidhybridization to form a hybrid between the primer and the target nucleicacid strand. A primer can be extended along the target nucleic acidmolecule by a polymerase enzyme. Therefore, primers can be used toamplify a target nucleic acid molecule.

The specificity of a primer increases with its length. Thus, forexample, a primer that includes 30 consecutive nucleotides will annealto a target sequence with a higher specificity than a correspondingprimer of only 15 nucleotides. Thus, to obtain greater specificity,probes and primers can be selected that include at least 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70 or more consecutive nucleotides. Inparticular examples, a primer is at least 15 nucleotides in length, suchas at least 15 contiguous nucleotides complementary to a target nucleicacid molecule. Particular lengths of primers that can be used topractice the methods of the present disclosure include primers having atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 45, at least50, at least 55, at least 60, at least 65, at least 70, or morecontiguous nucleotides complementary to the target nucleic acid moleculeto be amplified, such as a primer of 15-70 nucleotides, 15-60nucleotides, 15-50 nucleotides, or 15-30 nucleotides.

Primer pairs can be used for amplification of a nucleic acid sequence,for example, by PCR, real-time PCR, or other nucleic-acid amplificationmethods known in the art. An “upstream” or “forward” primer is a primer5′ to a reference point on a nucleic acid sequence. A “downstream” or“reverse” primer is a primer 3′ to a reference point on a nucleic acidsequence. In general, at least one forward and one reverse primer areincluded in an amplification reaction.

Nucleic acid probes and primers can be readily prepared based on thenucleic acid molecules provided herein. It is also appropriate togenerate probes and primers based on fragments or portions of thesedisclosed nucleic acid molecules, for instance regions that encompassthe identified polymorphisms of interest. PCR primer pairs can bederived from a known sequence by using computer programs intended forthat purpose such as Primer (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.) or PRIMER EXPRESS® Software(Applied Biosystems, AB, Foster City, Calif.).

Sample: A sample, such as a biological sample, is a sample obtained froma subject. As used herein, biological samples include all clinicalsamples useful for detection of fetal aneuploidy, including, but notlimited to, cells, tissues, and bodily fluids, such as: blood;derivatives and fractions of blood, such as serum; urine; sputum; or CVSsamples. In a particular example, a sample includes blood obtained froma human subject, such as whole blood or serum.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in Wilms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods. This homology is more significant when the orthologous proteinsor cDNAs are derived from species which are more closely related (suchas human and mouse sequences), compared to species more distantlyrelated (such as human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. To compare two nucleic acid sequences,the options can be set as follows: -i is set to a file containing thefirst nucleic acid sequence to be compared (such as C:\seq1.txt); -j isset to a file containing the second nucleic acid sequence to be compared(such as C:\seq2.txt); -p is set to blastn; -o is set to any desiredfile name (such as C:\output.txt); -q is set to -1; -r is set to 2; andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two sequences: C:\B12seq -i c:\seq1.txt -jc:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2.

To compare two amino acid sequences, the options of B12seq can be set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (such as C:\seq1.txt); -j is set to a file containing thesecond amino acid sequence to be compared (such as C:\seq2.txt); -p isset to blastp; -o is set to any desired file name (such asC:\output.txt); and all other options are left at their default setting.For example, the following command can be used to generate an outputfile containing a comparison between two amino acid sequences: C:\B12seqc:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the twocompared sequences share homology, then the designated output file willpresent those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1154 nucleotidesis 75.0 percent identical to the test sequence (i.e.,1166÷1554*100=75.0). The percent sequence identity value is rounded tothe nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 arerounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 arerounded up to 75.2. The length value will always be an integer. Inanother example, a target sequence containing a 20-nucleotide regionthat aligns with 20 consecutive nucleotides from an identified sequenceas follows contains a region that shares 75 percent sequence identity tothat identified sequence (that is, 15÷20*100=75).

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions, as described above. Nucleic acid sequences that do not showa high degree of identity may nevertheless encode identical or similar(conserved) amino acid sequences, due to the degeneracy of the geneticcode. Changes in a nucleic acid sequence can be made using thisdegeneracy to produce multiple nucleic acid molecules that all encodesubstantially the same protein. Such homologous nucleic acid sequencescan, for example, possess at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% sequence identitydetermined by this method. An alternative (and not necessarilycumulative) indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid. One of skill in the art will appreciate that theparticular sequence identity ranges are provided for guidance only.

Short Tandem Repeat: a pattern of two or more nucleotides are repeatedand the repeated sequences are directly adjacent to each other. Thepattern can range in length from 2 to 16 base pairs (bp) (for example(CATG)_(n) in a genomic region) and is typically in the non-codingintron region. A short tandem repeat polymorphism (STRP) occurs whenhomologous STR loci differ in the number of repeats between individuals.By identifying repeats of a specific sequence at specific locations inthe genome, it is possible to create a genetic profile of an individual.There are currently over 10,000 published STR sequences in the humangenome.

Single nucleotide polymorphism (SNP): The polynucleotide sequencevariation present at a single nucleotide residue within differentalleles of the same genomic sequence. This variation may occur withinthe coding region or non-coding region (i.e., in the promoter region) oran intergenic (between genes) sequence of a genomic sequence. Detectionof one or more SNP allows differentiation of different alleles of asingle genomic sequence. All common SNPs have only two alleles.

SNPs within a coding sequence will not necessarily change the amino acidsequence of the protein that is produced, due to degeneracy of thegenetic code. A SNP in which both forms lead to the same polypeptidesequence is termed “synonymous” (sometimes called a silent mutation)—ifa different polypeptide sequence is produced they are “nonsynonymous”. Anonsynonymous change may either be missense or “nonsense”, where amissense change results in a different amino acid, while a nonsensechange results in a premature stop codon.

Standard control: A value reflective of the ratio, or the amount orconcentration of a fetal genomic sequence located on a chromosomerelevant to a particular chromosomal aneuploidy (such as trisomy 13, 18,or 21) over the amount or concentration of a fetal genetic markerlocated on a reference chromosome, as the amounts or concentrations arefound in a biological sample (for example, blood, plasma, or serum) froman average, healthy pregnant woman carrying a chromosomally normalfetus. A “standard control” can be determined differently and representdifferent value depending on the context in which it is used. Forinstance, when used in an epigenetic-genetic dosage method where anepigenetic marker is measured against a genetic marker, the “standardcontrol” is a value reflective of the ratio, or the amount orconcentration of a fetal genomic sequence located on a chromosomerelevant to a particular chromosomal aneuploidy (for example, trisomy13, 18, or 21) over the amount or concentration of a fetal geneticmarker located on a reference chromosome, as the amounts orconcentrations are found in a biological sample (such as blood, plasma,or serum) from an average, healthy pregnant woman carrying achromosomally normal fetus. In some embodiments, a standard control isdetermined based on an average healthy pregnant woman at a certaingestational age.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals (such as laboratory or veterinarysubjects).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Assessing Fetal Aneuploidy Using a CpG-Containing Genomic Sequence

The present methods utilize an assessment of the methylation status of aCpG-containing genomic sequence in fetal DNA, in order to detect a fetalanuploidy. Basic texts disclosing the general methods of use includeSambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed.2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994), which are incorporated herein by reference. Any one of thesequences identified herein, including SEQ ID NO: 1-68 and 83-85 andportions thereof, are examples of sequences that can be used to detectfetal aneuploidy.

In some embodiments, the methods include (a) selectively purifying fetalDNA from a maternal biological sample using the methylation status of aCpG containing genomic sequence, wherein the CpG-containing genomicsequence is at least 15 nucleotides in length, comprises at least oneCpG dinucleotide, and is within a region on chromosome 13, 18 or 21. Insome examples, the CpG-containing genomic sequence comprises at least 15nucleotides of a nucleic acid sequence set forth as any one of SEQ IDNOs: 1-68. The fetus is genotyped using the purified fetal DNA to detectaneuploidy in the fetus. The methods can include the use of more thanone of the CpG-containing genomic sequences. Thus, the method can in theuse of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65 or all of SEQ ID NO: 1-68 and/or SEQ ID NO: 83 (or a portionthereof). Thus, the methods can utilize at least 15, at least 20, atleast 30, at least 40, at least 50 or at least 60 nucleotides of one ormore of SEQ ID NOs: 1-68 and 83.

In some embodiments, the method comprises (a) determining in abiological sample taken from the pregnant woman the amount of amethylation of a CpG containing DNA of fetal origin, wherein the CpGcontaining DNA is located on a chromosome relevant to the chromosomalaneuploidy or within a section of a chromosome relevant to thechromosomal aneuploidy, and wherein the methylation of the CpGcontaining DNA of fetal origin is distinguished from its counterpart CpGcontaining DNA of maternal origin due to differential DNA methylation;(b) determining the amount of a genetic marker of fetal origin in thesample, wherein the genetic marker is located on a reference chromosome,and wherein the genetic marker of fetal origin is distinguished from itscounterpart of maternal origin in the sample due to difference inpolynucleotide sequence, or the genetic marker does not exist in thematernal genome; (c) determining the ratio of the amounts from (a) and(b); and (d) comparing the ratio with a standard control, wherein theratio higher or lower than the standard control indicates the presenceof the chromosomal aneuploidy in the fetus. Typically, the standardcontrol value approximates the expected gene or chromosome dosage orratio in the human genome, although slight variations may existdepending on the specific methodology used in the detection method. Insome cases, the sample is maternal whole blood, serum, plasma, urine,amniotic fluid, genital tract lavage fluid, placental-tissue sample,chorionic villus sample, or a sample containing fetal cells isolatedfrom maternal blood. In other cases, the sample is any sample thatcontains fetal nucleic acids. In some embodiments, step (a) includestreating the sample with a reagent that differentially modifiesmethylated and unmethylated DNA. Such reagent may comprise bisulfite ora protein or chemical that binds to DNA based on methylation status; orthe reagent may comprise a restriction enzyme that either preferentiallycleaves methylated DNA or preferentially cleaves unmethylated DNA. Insome embodiments, more than one methylation marker or more than onegenetic marker may be used. Thus, in some specific non-limitingexamples, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 geneticmarkers can be utilized.

Fetal DNA co-exists with maternal DNA in the acellular portion of apregnant woman's blood (for example, serum or plasma). DNA from fetalorigin and maternal origin can be distinguished to ensure accurateresults in fetal DNA-based diagnosis. U.S. Published Patent ApplicationNo. 2003/0044388, incorporated herein by reference discloses that fetaland maternal DNA can be distinguished by their different methylationprofiles. U.S. Published Patent Application No. 2003/0211522,incorporated herein by reference provides methods wherein methylationmarkers can be used for prenatal diagnosis.

In additional embodiments, step (a) or step (b) may include the processof amplification of the methylation marker and/or the genetic marker,especially the methylation marker and the genetic marker of the fetalorigin. As one example, the amplification is by a polymerase chainreaction (PCR), such as a methylation-specific PCR; or the amplificationmay be a nucleic acid sequence-specific amplification.

Fetal DNA can be hypomethylated relative to adult DNA reflectingtranscriptional silencing of specific genes expressed early indevelopment. One means of generating fetal-specific PCR products is toidentify loci that are unmethylated in fetal DNA and methylated inadult/maternal DNA. Another means to detect fetal-specific DNA is toidentify loci that are methylated in fetal DNA and unmethylated inadult/maternal DNA. Loci of this type are differentially reactive withbisulfite such that unmethylated Cs in DNA undergo oxidativedeamination, resulting in C to U transitions. Methylated Cs are notreactive with bisulfite, and consequently, are unaffected. Bisulfitetreatment of fetal and maternal DNA present in maternal serum willcreate primary sequence differences between fetal and maternal loci thatexhibit differential methylation. However, restriction enzymes thatdifferentially recognize and clear unmethylated DNA can also be used. Inother embodiments, the method for selective enrichment of fetal DNArequires the use of the methyl-CpG binding domain of human MBD2 protein,which is coupled to paramagnetic beads, for example DYNABEADS® M-280Streptavidin, via a biotin linker. Without being bound by theory, thehigh affinity of the MBD-biotin protein for CpG-methylated DNA providesgreater sensitivity than antibody binding, while the use of theDYNABEADS® provides a simplified, streamlined workflow.

In one embodiment, the DNA is amplified using quantitative PCR andprimers selected to amplify sequences on a potentially abnormalchromosome. Control quantitative PCR with a second pre-selected primeris conducted on a normal or control chromosome (i.e., a chromosome nothaving the suspected anomaly) and the ratio of the quantity of the twoPCR products are determined, thereby detecting fetal aneuploidies. Ifthe loci of interest are from chromosome 13, 18 or 21, and quantitativePCR strategies are employed. In some embodiments, real-time PCR isutilized and chromosome copy number are determined. If the loci are alsohighly polymorphic such that both alleles can be discerned, chromosomeaneuploidy can be readily revealed. Other amplification methods can alsobe used, such as Loop Mediated Isothermal Amplification (LAMP).

In some embodiments, methods are provided for detecting fetal chromosomeaneuploidies by treating DNA isolated from maternal serum with bisulfiteor restriction enzymes and then performing quantitative PCR on thesample with a primer pair homologous to a test chromosome sequence thatis differentially methylated in maternal DNA and in fetal DNA, where theprimer pair only primes bisulfite treated unmethylated DNA orrestriction enzyme treated unmethylated DNA. A “control” quantitativePCR is conducted with a primer pair homologous to a control chromosomesequence that is differentially methylated in maternal DNA and in fetalDNA, where the primer pair only primes bisulfate treated unmethylatedDNA or restriction enzyme treated unmethylated DNA. The ratio of thequantity of PCR product produced for the test chromosome is comparedwith the control chromosome, thereby detecting fetal aneuploidies.

In some examples, the mother and the fetus are human. However, themethods can be used in other mammals, such as, but not limited to,non-human primates. In some examples, the subject is human and the fetusis between about 10 and about 14 weeks of age, such as between about 11and about 13 weeks of age. Thus, the fetus can be 10-14 weeks of age,11-13 weeks of age, or 10, 11, 12, 13 or 14 weeks of age. However, thefetus can be of any age. In some examples, the mother is in the first orsecond trimester of pregnancy.

The methods disclosed herein can utilize the following nucleic acidsequences, or a CpG containing nucleic acid sequence within 100kilobases (kb), 50 kb, 25 kb, 10 kb, 5 kb or 1 kb of these sequences.

In some embodiments, the methods disclosed herein utilize at least 15,at least 20, at least 30, at least 40, at least 50 or at least 60nucleotides or all nucleotides of at least one of the following nucleicacid sequences:

SEQ ID NO: 1 ACAATATGGAAAACACAGCTGTAACCCATACACATTCACACCAGCACAAGCTTCTTCCATCCTTCCTGGAGAGAGACAAAAACAGAAGACACAGAAAGGAAGGGCAGGATCAGGGACAGCAGGTTCGTGGGAGGGACTGTTCGTGGGGGA ACC

GCAGGGGAAGAAAGACAGGAAGGGGAAGAAGGGGAAAGAGAGGAAGTGGGGTAGGGCAGAGGGCCCTGGGCTGTGCACTCCCATCTCACACTTGTCCCCAGAGCAAGGTCTTTAAGGCCAGACTCCAACCCAGGAGCAGGGAGGGGGGCAAACGCCCCTGTAGCCGCCCTGCACCCTCCCAGGAGGGGGAGC ACAGGCTGTCSEQ ID NO: 2 CCACAACCTGTCAGAAATGCCCCCAAGCCCAAAGGCGTCGAGAGAATGGCCAGGTTGTTTCAGATTGACACATATCCTAATGTACAAGTCAGCCCACACACCCCACGTGCACTGAGCGTCTCTTGTTGTTCACCCCAAATAAACTCTG C

GAACTGGGGCGGGACTCGCAGGGGCGGAGAAGGGGGGAGACGGGCAGAGGGCAGAAGTGGATGGTGAGAAGAGCCAATGGAGGGGCCCCGTGAG AGTGAGCAAGGCTGCACCCCTSEQ ID NO: 3 TGATAACACTGGGGAGAGTTAACTTTTTCACCTCTGTTTGTTGATATGCCCCTTTGTGCTCCGTATTCACAAAGGGGCACCTCAACAAGCCCATCATTTGTTAATGAATGCCTTTACTTTCATCGCCAGCAAGACATTTTCATAAACCCACCATCTACTTTGCAGTTCTCAAGAGTTGCTTCTCTTAAACTGGTGAGGACGCGGCTAAGCCCTGGAAATGAGTCCAGATTCTCACGGTGGCCCATAAACACTGCTGTTTCCTCCCACCTAGGAAACCCGCAGACCTGCAGAACCTGGCTCCCGGAACAAACCCTCCTTTCATGACTTTTGATGGTGAAGTCAAGACGGATGTGAATAAGATCGAGGAGTTCTTAGAGGAGAAATTAGCTCCCCCGAGGTAGGCCTCAGAAAACCAGTGTTCATAACTTGATTGTCACTTTCCCCCACTAGTAGTCAGTTCTAAAGCTCTGGGCAGTGTGTGTGTGTGTGGTTTTCTGCAGCCCACGGTGCTCACTTCCTTAATCATAACCCTGGTGTAACCAGATTAGAGTTCGGGGACCTGGGTTTCATTGTGCTGCACCTGCAGCT TGGCAATCACAGTSEQ ID NO: 68 CCTTTGTTCTTGTTGGCATCTGTGTAAGAGAATCCAGGGCCTGAACCATTGTCCACTCAAACAGACCATACACACCTGGTCCAGTTTTGTGCATGCCTCCCTTTTCCACAGTGTGCCACTTGCTATAGTTCTGAACAAAAACTCCCTTGCCCTTCGGAGCTTTATTTATTTTTAATTTGCTCTGTCATTGCATTGCATGCAATAGAAGCTTCCTGGTGGAAGCTCAATGTTCTGCTCAGTCCCAGACACGTTTTCAGCCACTGTATCATTGCCTTAGGTTGTGGTTTCCCCAAAGCAGCACCGGAGGTAAGGGCTTGTGTGCAGGAGTTCACTTGGGAGGTGGCTCTAGGAAATAGAAGCGAGTTTCCAGGAAGTGTGGGATGAATACGGGGATGGGGTGGGGGGCGGGGAGGAATCCAATATGAATATATAATCTCATATTGAGTATAAAAATCCAATATGAGTATGTTTTCCAGACTGCTGCTAAGGAGAATGGTGAATTATTCTACTGCGACCTTTTGAGGGTCCACACAATGCCTCCCAAAACTATCTACCAGAGGGTGGATAGGAAGCATTGATCCATGGCTT ATATTCCCCATTGSEQ ID NO: 4 ATCAGAGTCGCCCTTGACCTCAACCCTGCCCGAGAAGGAAGTGCTGGTGCAACCCCAGCCCAGCTGCAGGAAGTGGTACCTGTCACCAAGTCCCCTGCACAGGGGAAGCAGGGGGCTGAGGGGGCAGGACCAGGGGGACGAGGTCATCCTTCCCCGGAGAACCCCTCAGGGGCCGGGCGCCCTTCCCTCCCGGGCACCAAATGGACACAGCTAACAAGAACCACACTTCACGGTGAGCAAAGCTCTGAACATTCAGGCTCTGAGACACGAAAGCGGCGTCAGAAGAGACCTGCAGCAGATCCGGGCCAAATCACACACACCCTTTCACAGAAGTCGAGGACAGGAGAACAGGAGCAAACACAGGAAAGGAAGAGGCCAGCCTGGTGGCTGACGCCTGCAATCCCAGTGTTTTGAGAGGCCAAGGCAGGAGAATCACTTGAGGCCAGGAGTTTGAGAGCAGCCTGGGCAACACAGTGAGACTCCTACCTCTACAAAAAATAAAAATAGAAAATAAATTAGCCAGACATGGTGGCATGCATCTCTAGTCCCAGTTACTCAGGAGGCTGAGGCAGGAGGATCACTCGCGCC CAGGAGGGAGAGASEQ ID NO: 5 CTCTGAGATGCGTCGTGTAGTAAACTTCTCAATAAGGCGTAGTTTATGTTCAAGAGAGAAATTCTCTTTGTCACTTAGCATAAACCTTGTAGCTAGCTTTCTGAATATTTGTATCTCAGTAGAAGAGTTAAATGCCTCATTCAAGTATGTTTTCAAAATAAAAATTGTAAATAAAATCACGTGATGTTTATATATGAGAAGTCACCAGCTTTTCATTCAGAGTTCCCCCGTTTCTAGAGGTTGGACCAGCCTCAATGATCGTGGGGAACCTGGTTGCTGGGAAGAGAATCGCTCAGGCTTCCGGGAGAGAGCTCGCCCACCTGGAGGACAGCGACCAGGCACGGAAGGTGACAGGCCAGTTCCGGGGACGGGTGGACGGGTGTCTGTGCCCCGGAGGCGCCAGTGAGCAAGTCGCCCTCTCGTGCAGTTCCTGTTCCTGGCTGACGTGCTGCAGTGGCGTGCCCACACCACTCCTGACCACCCGCTGTTCTTGCTGCTGAACGCCAAGGTGAGGCAGTGTCACGCCCACGGGGCTTGGAAACACCTGTGGGCCGGTGGAGCCCTTTGCTTGTCTAGTTCATGGTGCCA GTGTCCTGGTTGTSEQ ID NO: 6 AAAATCTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTCGCTTTTGTTGCCCAGGCTGGAGCGAAGTCTCTTAGTTTCCTCCCCACGGTTTCGTGCACTAGACCCCGAAGCAGGCAAGCTGAGCATCTTTATCCCCATTTTTCAGAGGAATGACTAGAAGTCAGGGTTTGCCCAAGGTCAAACAACTAGCTGGTTGCAAAGTAAGAATTGGAAGCCACCCATGTTATTCCTTCCGCTACACCTCCCTCCAGAGACAGGGCTGGCCAGCATCGCACGGAACAGGAAATTGAAATGGCTCCCGGTAAGGCAGCGAGCCTGCCAACCACCGAGAGAAGCTGACAAGTGACCCAAACCCGCCCAGAATTTAAAATGCCTCCTCAGAGGAGTTGCCGACTAACCTCCTCCCACCTGCTGCAGTGAAGAAATAAGTTCCAAGAGTCTGCAACCCACCATGCCTGTTTTTCCACGGGAAGGACCAAGTTAGAAAGGAAGCTTGAATAAGTGGGATGCTGTTGAAGAACTAAGATGTAGAAGCAGTTTCCCAATATGAACTTACTAGCTTTTTTCCCACTCCTATCACATCGCC ACTCCCCCCACACSEQ ID NO: 7 GCTCCTTTTTGGGTAAGGTCTGTTGCTTCTCTAGGAACAGTGACGGTGGCAGAGCCCGTGGCCCCTCTCTCCTGTCCCAGAGCCAAGCTGTTTCCTCTCCCCACTCCCGGGCACCCTGCGGGCAAGTGAGGGGGGCCCACTCTGCCCTGGCCTGCCCCACCGCCCCATTTGCTGGGTTGTCCTCAAAGCACACGGTAAGCACCCCCGCCCCCGCCCCCCACACACGTACCGCTGTCAGCCTTCTTGCTCGTCACCTCCATCCAGGGCACATCCGCCCCTCTGGGTGCTGTGAGCCTGTTGCCGGCGCGGTCCCGCTGCTCCTCCTTCTGCTGCCCTGCACTGCCGTGGCACAACCCTCCCTCCGGGTCTCTGCTGAGCCCTCCAGGCGCTCAGGTCTGCCGTCTGGTTGCAGGTGTTTTATAGAGTGCAGCTTTACGCTCCCACTCCAGTCATCTCCACGTTGGTGTCCGTCTGTGTGTCCTCTCTGCCTTTAAGTTCTGCCTCTGTGTGACACAGTTTCACAGTTTCTGTGACCAAAACACAGGTTTAGTCGCTTGCTGCTGGCAGAGTCCAATTAACAAGAGCACG TCGGGTATAAAGASEQ ID NO: 66 CAGAGACGAGAGACACTTTCTTTGTACATGTCCCACAAGCACAGTTCCAGGATTTTGGCACTAAGTAAACAAATAAAAATATGCAACAGACATAAGAGATAATAGTAATGTGTATTGAGAAATATAATTTTATATAGTTGTTCCTTTTTCCCAAGATTCTTTATTAATATCACCAATAGTTATTACTAGAAAACACTTTCAGAGCTAGAAAGTTGCCAGAAACATTTCTAATTGACTACTGTTATAATTAATATTTTGAAATGCTGAGACATTTGCTAGGGTGGACAGCTCCTTCAAAACCCGGCCGTTATCTGACGAAAGAGCCTAGGCTCAAATAAGAATTAAACACAAGTTGGAAGTAAGGTGAGGCACAGTGGAATTTTTTTAGGATGGATATAGTTTTCAATAACCTTAATTTGGAATTTTTCCTTTGGGATAATGACTTTGAGTCTATAGAAATATTGTATGCATTATAGACGAATTGATTTTTTAACTAAATTCTGAAAGGCTTAAGTAAAATGATTTGATGATCAAGTCAGATGCATTTCTAAGGAAGTGAGGATCACATTAGACAGAAAGTTTGGGTTT GGTGTATAATAATSEQ ID NO: 83 CAAATTCTTCCCAAGTACTGTTTCCTTTCCTCAATTCCTGGCCTAGCAAGTCTAGAAAGAAAACAAAAAGAATGTAAAATGTTCTTGGTTGACTTTACTCTTAATTACTAAAAAGCAAACAGAGAAAATTAAAATCAAACACATGCAAAAAATTCTTCTAGAGCCTTGCACACAATGCCAGTTCTTGAAATATTTCATAAAGCAAAGCTAATGAAATAAACAAAGACGTGGCTGAAAGGATATGCATTGTACTGTGCTTATGGAGCCACATTGCTCACTGCATCTCTCGCTCCACTGCCGCCGGGCTCATCTGCCAGAAGCCTGTTCCTGAGCTGATTGGGGTTCTGGTCCAGGGGTCCCAAGGAGCGAGGCCCAAGCTCAGGAGGACAAAGAACGACCACCTGGAAAGTGGCAAAAATCTGTAAAAAAATAAAATCTGAGTGGGCCCAAGGGGCACCTGGTCTCAAGAGCAAACGCTGTGTCCAGAGCCTCATGTCCGGAGGCCCAGCCCGACTCCCTGGCTCTTACAGCCCCGAAGCCCAGAGACCACCCCAGCCCGACTCCCTGGCTGAGACTTCCCGCCCGGAT GCCGTGAGATCGTSEQ ID NO: 8 ATGTGAGAACCCAGCACCAAAACTGGGCTCACGGATCAACAAAAATAACCCGGGCCTCACCGACCAAAATCAAGTAGAATAGCGGTTGGGTCAAAGGAGAGAAAAATAGGCTTGGTTGCTAACCIn some embodiments, the methods utilize one or more of SEQ ID 1-7, 66,68 or 83. In additional embodiments, the methods utilize all of SEQ IDNO: 1-7, 66, 68 and 83.

These nucleic acid sequences can be used to purify fetal DNA and/ordetect aneupolidy of chromosome 21 (SEQ ID NOs: 1-7, 66, 68 and 83) orchromosome 18 (SEQ ID NO: 8). In some embodiments, the methods disclosedherein utilize at least 15, at least 20, at least 30, at least 40, atleast 50 or at least 60 nucleotides of these nucleic acid sequences.

The following nucleic acid sequences are less methylated in fetal DNAthan maternal DNA, and can be used to detect fetal aneuploidy ofchromosome 13. In some embodiments, the methods disclosed herein utilizeat least 15, at least 20, at least 30, at least 40, at least 50 or atleast 60 nucleotides or all nucleotides of at least one of the followingnucleic acid sequences:

SEQ ID NO: 9 TAAATATCTGTGAGAAATATTTGTGAAAGTATATTATGGAAAAAATGGAATATGCATGTAAACTTAGGCTATGTTGAGTAAAATAAAACTAATGTTTTAAATATGAAGAAATGAACAGTTGCTCAATGCTTGTTTCTCTACACATGTGCAGGAAGTATATAATTCAGTGCTGTACACATATCACATTGGAGCATTAATCACAGCCGTGTCCCACTTTACCCAAACATGGCCACATAGTCTCTTAATCCAAGCATTTGGGAAACTACTAGCAATCATTTAGAGTGGAAACATAATTTGACACCGGTTTAGCATTCCATTTGTTTGCTACATCCCTCTTTATGACAAGAAAGGGAAAAATCATGTAATACTATAATTTGAGGAAATGTAATAATGCATTCAGGGAGACAGAAGCAGGAAAATTTTGCCTGTGTAGAATATTGTCACACATACATGTATGTGTATGTGTATGTATATATCAGAAAACATATTTCAAAAATAAAAAAGCTATGCTTAGTATACCCCATTGCAAAATCACTTCATAATGAAAGCACAGACCATGTACAACAAATAAGTGTGACAGAAATGAGT TTGGCAACTACCCSEQ ID NO: 10 ATTTACAACAGCAATAGGTAGCAATAGGTCTCATGACTTTACTTCTCTAATGGATTGAAGAAAAACCAGTGAATTTTAATTTGTCCTGCCTTTGCTTTTTGTGAAATGAGAACAACTTACAAGCCCTTCAGATATTAGAGCTAATACTAGAAATCCTCTGGTGAGGTTTTTATTACTTATGTTATATTTTACAATCACAAATTCTCTATTTGCCTCTTTTAAAAAAATGTTTTCTAGGTGGGAGTCGGTGGCTCAGGTAAGAGATACTGCCTCATGTAACTCTCTGGGTATGACTTTCTCCCGGATTTAAGGGTAGCAATAGGTCTCATGACTTCAGTTCTCTAATGGATTGAAGAAAAACCAGTGAATTTTAAATTGTCCTGCCTTTACTTTTTGTGAAACGAGAACAACTTACAAGCCCTTCAGATGTTGGAGCTAAAACTAGAAATCCTCTGGTGAGATTTTTATTACTTCAGTTATATTTTACAATCACAAATTCTCTATTTGCCTCTTTTAAAAAATATTTTCTAGGTGGGAGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGTGCGG ATCACCTGCCTGASEQ ID NO: 11 TTCTATTCCTTTCCTTATAAATCCAAGCAATAGTTTCATTCTTTAAAAATGCATAATGTGAAAATAGCATTAGGAAAGCTGCCTTGAAATATGGATACTAAATGCATCCCACTGGGCAACTTCAGCAAACCGCTGGGTCTTCCACCACAGTTAAAATAGCTCCTGCTGTCCCCGGGGCAGGAGTGGCTTAAGAAAGCATTTAATCCAGGAGTGCGTGTCTGGAGCTTTGTGTTAATCAGATTCCATATGACTGAATGACCTTTGTTTTCCCTCTGGTGGAATCGTGAACATTTGGGAAAACCGGTTTGCTTTTAGTGAGTGTTGCAGGGTGCAAATGAAACTGTATTCAACCTAGGATTCGACTGGGAGAAAAACAGGAGAAAGGCCACAGGATTGGTGCAAGGACAGCAGGTCAGTGTCAGGGGCTAGGTGACTGGGGAGAAAGCTTGCTGTCCCCTCTGAGTGTCCTCCCCCAAACCTCAGCGTCCCCCCTTGGGAACCCATACCATGGCCCCGGGCAACAGAGGCAGCAACACAAATAAATAGAAGAACTGTTTCATTAAAACCTCCTCCAGACAAGGCCAGCTC TGGATGCAGTCTTSEQ ID NO: 12 GATGGTGAACTGTGTTCTGGCGGACTTATTTTTAATATACCAATTGTGTGTAGCACAATAGTCATTTCATAAAAGGTCTTCAATGTGTTGTAATCACTGAATGGGAAATAAATTTCCATCCTCTTTTGAAGGGTGCATTTGTGATGTTATACTTGCTGCATATTGTGGTAACAAGCAGTCATTCCCAAAAATGAACATGACCCTATTTCATAACAGGATATTGATTTTGCATGCTTAAATTAAGTTTGTATTTATTATTACTATATATCTCTCCAGTTAGTTTCCATTTTTACTTCTAATCCGGTTAAAAACTTACTATATTTTAAAAATACTCTCTGCTATAGCCATCTAATTATTTAAAATAGAATCTCAGGTTATTAGGCAGTGCATTGCTTCCTACATCCAAATTTGAGCTTGAACATTTTGCTAACTCTTTTGGAATTCAATTTCCAGTTTACTCACCTCTGAAGAAGTATGGATTACTTGACTTGTTCACTAACTTTAGTAGAATTAGAACTCACACTCATGTGTCCTAACATCAAATTTTCATTGAAGATCATGTGCTGCATGACCTATACAGATGGTCCC CAACTTGAGATGGSEQ ID NO: 13 TTTGCAAAGAATTCTCTAAGAAAGACATTTTCCTGCTTTCGTTTCTGTTTCATGCTGGGTAATGTTGGCAAGAATTATTCATGGCACATGTGAGCAATTCAAAGAAATATTGTAAAAAAAAAAAAAAAAACAAGGCAGCTTTGCAGAGGTGCGCTATCTGCTTGATGACTTGGCATGCTGCCTCTGGTGCTTGGCATAGCACCCTAGCTGCTGGCACAGGGGAAGTTACATATACTTGCCATTGGGCCTCTACTATGCTACCTGCTGTGCACGATCAATATCAACCAACTGCATAAACAACCGGATGAACCTTGTATCTGAGGGACAGCAAATAGCATTTGAATTATTTTACATAAATCTGAAAATGTAATAGCGGTTGATGTTCCTATTAGTATTATATCTGAGCACGATGATCATAATTGAGTAACATTAAATTTAGTTTATTGAACAGCAGTCCACTGCCCGGTTTACTGTGACTACGATCTGTGTAATGAAACCAGCAATATGAAAAATTCTCTCAGTTGGTACACTTCCTGGAGAGGAGCCAAAGTATTTATTGCTATTTGTGCAATTAATGGCCGTATCGAT TTCTTTTTTTTCTSEQ ID NO: 14 TTACCAGGACCTCACTATAGGAAGAAAAAAAAAGTAAAGCAACCTCGCAGGTATCTTTGCTGGTTAACATCATAGAGTATGAAGGGTATCTTGATGTGATCCATTTTAAGCTACAAAGTTTCTTACAGTCATACTCAAAACTGGCAAAATACAGGACATCATACACATTTTCATATTTTATTCTAATGTTTATTCTGAGGTCAACAATATAAATTAGTTTCTGAGATGCGCAGATAAATTAAAATTCCATGCAGGTGACCATAGACATACCCCTACTATGTGTATCTGTCTAAACTATAACCGGAGAGGACTACAATTAATCCCTTAAGCCCTTTATCTTCTTTTCGTCTCCTGCTTGGTTCTACGTAAACACCTTTGCTGAACTGGAAGATCCAACCAACTTATACGAGCTTCTTGGATGCGAATGAAAACCTCGTGAATTGCCCAGCAAGTTGACATTCCTTTCTAGATCCAGCACATACAACTCTCTCCAGGTGAGCACAAAGAAAGAAGAGATTTGAAGTCAGCGAGGTGCGTTTTATTGGTTCAAAAGGGGAGGTTATTTTTTAAAGGCTTATGGAAAAAGGT AGCTATGTTTGTTSEQ ID NO: 15 TTTGTTAGAAAGTGGCTGTGATCACAAATCAAAAAATACTAACTCATGTACAAGTGAAGGACCACTGGCCACTTTGGAGGTCTCTTGATCACTCTTGGATTCCCATCAACCTTAAATAGGATACCAGTCAGTCCCCTGCTGCCTATCCTCAAGAATTCTTTCACTTTGAAGGATGTATAACACCCATAACAACTTATGAGGCAAACATGTTATACATGAAGGCAACTTTTCCAATATATCACCTCCCCCCATACAACTTAACACATCTGAACTTTCATGGGATTCAAAATACAAAAAAGTCCGGATTCTTTCATATGCACGCAGATCCAATAATAACAAATAATTAATGATTCTGAGTGTTATTTTATGAGGATGGCATTTTATATCCCTCATGCTTAGGGCAATTCTAGAAGGAGATATTATCACCATTTCACTGTTGAAACTGACTCAGAAGCTTATCCAAGAGCACAAATCAACATTAAGTAACAGAGCTGGGATTTAAACGCTGCTGAGAAATCCGAGAGGCTCTGTATTTTTTAAAAACAACTTTTAGAGTAACCTTTTCTTTTCAGCCATGTTAATTATAGG AAGAGACTACAATSEQ ID NO: 16 CGAGAGTCTGGCTTCCAGTGAAGAACACTTGACAAGGGAGAAAATCTGGACATTTCTGCCTTTGAGTTTTGCTTAGGCCAATCTTGCAATTCTCCCTCAATCCACGTGGTCACCATCTTCCCACCTAAAGAGCACTCACCTCCTTTGAGAAGCCTGTGCTTGTCCTGACCCCTCTGAGCACACTTTAAAGTTTATGAATTCATTCCTTCCTTTGCCATTTCCAGAAGGTCTTTGTATTTTGCACAAGGACTCTAGTTCTTTGTGCTGGCATCAATTAGAGACTGTTTGTTTTCTGTTTTCCCGGGTTAATGGTAAATGTTTTAAGGGTAAAGATCTTATCTTTACATAGCATTGATTTTTGAACATTTGATTACTTGCTTATTTATTAAGCCTTTGCTCATTTGCCTTACCATAATCTTTTCTATACCTTCATGTTTGAATTGTTTTTATGGCTTAAGTATATACATATAATATGTATACATAATTATATAAAATATGTATATTCTCTTTTATACACTATGAAAACAATTCTTGGAAGCACATAAAATGTCTTTGCACAATAAAGAAATATTTTAATATTGATAATTC ATAAATTATATTGSEQ ID NO: 17 TGCTCACTTTTTCCTTAATGGAGATTTTGTTCCATGTATGGTAGGTAATGATCTGAGGCAGGAAGACAAAGAGCAGATAGCATTCAGGGCCTCAAAAGATTCCCCTAATGTGTACAAACATACTAGATCTGAAGTTCTGGTTCACCATGGTTCTTCCTCATTAAGCCGCAGATTTCCACTTGGAAAATTCATTGGTAAAGTGCCATGCCTGCTTGCTGCTGATAGGATTTTCTTATTGTCCAGCAGTATGCAAGTATTCAGCCCTCAGTCTCTCAGAAGAGGGGTCTGAATTCCCTTGTTCCGGGGTTGTGACCTTAACCTTGTCCTGAGATTTATAGCCCCAGAGAGGCTCTCCTCCTGATTTACATAGTTCCTGATGTCACTTTGAGCAGATTTAGTTCCAATCTCTGCAGAACTCTTGACTATGGAATGTGGGAACACGACGAGCTGTTTGTTTTATACTCCCAAATCCTTGAACAAAAGTTACAGAGTGTTTTTCTATCTTCTCAGAGGAAGAAGGCCCCTCTCCCATTCCCATTCAGTTCTACCAAGCAGCAGCTGTTGCTGTGTGTACTGTGTTTAAAATCC CTTTGTTTTATGCSEQ ID NO: 18 TTCTCTTTCTTAATGATAAAATTACCATAGTACCAAAGTAACTTAGCTTTAGCCTTCTTCTAAAATAGGCATATTCAACCTCAGCAACTGGGCCAGACAAATTTTTGTTGAGGGAAATGTTCTTTGTGTTTTAGGATATTTAGAAGCACTTCTGGCCTCTGGCCAATAGCTGCCTAACACTCCTCTCCCTCTTTTCTAGAGTTGCAATAAAAAAATATGTCTAGATGTTGTCAAATCTTTCTTGGGGGGCAAAAGTGTCACCTCTTCAGAACTACCAGTTAGTAGATATGGTAAAAAGAGCCGGGTTGCTTTTTAATTTTATTGCATATTGACAAATTATAATTGTATATAATTATGGGATACAAAGTGCAGGAGGCTGAATGAAATAGGAGGGATCACCTGAGTCTGGGAGTTCGAGCTGCAGTGAGCTATGATCGCACAGTGCACTCTAGCCTGGATGACAGAGTGAGACCCTGTCTCAAAAAAAAAAAAAAAAAGGATAAAAAAGGAATATGCGATAATTGCGTACCTGCGATAATATGATGCAGGTACGCAATGTGGAATGATTAAATCAAGTTAATTAACATA TCCATCATCCATCIn some embodiments, the methods disclosed herein utilize at least 15,at least 20, at least 30, at least 40, at least 50 or at least 60nucleotides of these nucleic acid sequences.

In some embodiments, the methods utilize one or more of SEQ ID NO: 9-18.In additional embodiments, the methods utilize all of SEQ ID NO: 9-18 todetect an aneuploidy of chromosome 13.

The following nucleic acid sequences are less methylated in fetal DNAthan maternal DNA, and can be used to detect fetal aneuploidy ofchromosome 18, In some embodiments, the methods disclosed herein utilizeat least 15, at least 20, at least 30, at least 40, at least 50 or atleast 60 nucleotides or all nucleotides of at least one of the followingnucleic acid sequences:

SEQ ID NO: 19 TGCTCACATCCCAATGTCTTAGTGACTTTAGGAAATAAAGCAGCTCTGTAATTTTTTTTCTTACATCCTCGCTATTCAACACCATACTCAGTACATAAATCCAGATGGGATCCGGACAGTAAAATAAATCTGAATGACAGATAATGTGCTGTCCTCAAAAATGACTCTCCTCCTTTTCAACTGAAAGAAGACATTTGTTTATTTCAGTGAAAACGAGTTCATTATTATCTGTTCCTACTTCCTTGGGAGTGATTATTCTGTCAGATACATGCTAAGTGCAAATTTTCTGCATAGCAGAAACCGGGTCTTGAATCCAGGTGTTCTGATTCTGAGTTTATTTCTCTAAACATTGGTAATGACCATAGAAAGTCTATTTTAAAAGGAAACACAAACCAAAGACTAATAAAACTATATGCAACTAGAGAGAATTGTATCATGATTTAGATGAGCTCTGCTTTCTACCCTTCTCAATTTAAGTGGGGGTTCATGTTATCCCTTACTTTCATTGTCATTTTCCTTGGTCCAGTGTTCTCCTAGGTCTCCAAATGAAGCTATCACTTGCAGTCTGCAAAACACAACACTGCACTA ATTTAATTCTGTTSEQ ID NO: 20 AATGTTCCTTTTAGATTCCCTTCTTTCCTTTTCCTTCCTTCCTTCCTTCCTTTTCCTCCCCTCCCTCCCTCCCTCCCTCCCTTCTTTCCATGTTGTTCAGGTCACACTCTTCAGCTAGATTTGGGGGAGAAGTGGGCTCAGAAATGTTGGGCATGTGTGGCAGCACCTAAAACGTTATGGAAGCAGGGTGAGTCCCTGAGCATGAAGCAAAGGAGACATGTAAAGGAAGGAATATCCAGTTAGTATCTGAAGGTGGAACAAAATCATTTAGTTAGTTCTAGCCACACAATCTGGTTTTACCCGGATTGAATAGTTATATGACTCAGGAATCAGAACCCAAACGAATTAATAGGCAAGAAAAATGGCATCCCAGAATCTGTGAAAACTCATATTGATTGAATTTTAAGAACAAAAAAGGACCTTAGACGTGTCAAAATCAACTATTCCAAGTCCCTCTGTGGTATTAATGACATCCTGTTAAAACAAAAACAAAAACAAAAACTTTCAGCAACATCACTTCAAAATTATCCTTTTTTATTTTTGCATAATTTTAATTCATAGAATCTTTTATGAAAATATTTCTAAAAA CTTTCTGCACTTASEQ ID NO: 21 TGACCTCATATGGCAGCAATCTGACTGGTGATTGCACTGAGAGACAATGCACATGGCATAAGAACAGCTCCTTTACGCTCCTCTTGATGAAGAAGAACAGGTGGAATACTTCATATACTTTAGATATTGATGTCACTATAAGCAGCTTTGACTTTGAGTTCAGAAAGCAATTGATGGGAATGCAGGCTTCTAGAAGTATAGCTGTCACACAGATTTAGGGTCTGAAAGCAGTTTTCTGGTTTACCTTTTGGCACACAAACCTTCTCTGGTAAGGGACTGAACTTAATTGGAGTCTAAGATCCGGTAGAGAGAGCCATATTGATACTTATATGAATTCTAGAGTTTTGGTGAAAAAAAATCAAAGGTAAGTAAGTGCAAAATAAATTGTTTTGCAGGCATAAATATTCAAACATATAAATATATGTCAGGTATAAATATACATATTAATATGTGAATATATTATATATAAATATAGTCAGATATAAATATTTAATCATCATAGATTTAGACCGAAGGAGGACAACATAAAGAACATTACTCATTTCTTTGAGTTGCTGGGTATAGCTCAATGATGGTGGATCTTGGGTA CCAGAAAAAAATGSEQ ID NO: 22 TTTCACTAAACATTATGTTAAGTAATGTATGTAGGTTGATAGGTTTATATGGTACAGTAAGTCATTAATTTTAAACCTTCATATGAATTCAATCAAATCTTTCATGCTATCTCCTTAGACATTAGAATTATTTTAAATTTGTGTTTTGTTCTGTTTTGTTACTACAAACATTGCTAGGATAAACATTTCGTGCATACGTGCAGACGTTTCTACACAGTTTATTTATGGTGGCCACAAAGTCTCTATATCCTAATTAGTGACCTCTGTGTTCTATTATTAGCACAAATGTATATTTTCAAACCGGATCTTGTAGACAACTCCTTGGGAGTCATTAACAGGGAGGGTACCTGATAGTTTTCTTCAGATCATTATTATAAGCAGGGATATCATGCATGGAGAATTTATAGCTGCTCTATAAGTGGAAGGGCATTGTTCCATTATTACTAAATATTGCCAAAGTGATCTTCACATAGGTTGCTTCTATGTATATCCCAACAAGAAATACACGGAAGTTCTCATTACTCCAGATTCTTGACAAGAATTTATATTATCAGACTTTTAATTCTTTTACCAAATGAACAATAATGA TTATATATCTTGTSEQ ID NO: 23 TTTGCCAAAGAACTATGACTTTAGAATATGTGTTATTTACTCTTCCAAGATACAGCTGAATTGGAGGGGGATTGGGTTGTGCAGATGGCACTTTTTGCATGTCCCTTGCTCAGATAATTGGATTGTAGAAATAAAAGAAATAATCAGGATGTCAAATTTTAACATTCCTTTTACTCATGGATACTTTGGTGTTGCTCTTATTTCCATAGGCAGAGGGCAGATGATTTTCAGCCCCATAGAAATAGCAGAATGTGCAGGCTACTAACTTCGCTTCCTTGAACTAACATTATGATATCATCACCGGATTCCTCAGCACAACAGCCTGTTCTTAACTCAGTTCCGAATGTTTTCTCTCACTATCATCTCGATCACTTCCAAATGTTTTCTCTCACTATCTTCTTGATCAGGCCATCCCATTCTTAGGTTCTTACTGTGCCTTCTCACTTTGTCTTAAACAGATAAAGTCCTTCAGCTGATTACATTTCAAATGCGTCACCAGGTTGGCCAGAACGCGTCGGTTTCTTCCTATCTTGTTTCTTCCACTTTTTTAAATAAAATAGATTTTTCTAGGAGGACACTATTTAACTT TTCAGCACATATCSEQ ID NO: 24 TTGCTGACGATTCACATTTTATTAGTTTGGTTATGTTTTGTCCTTTTAAAACATTTTCTTTTGAGATGGGGGTCTTGCTCTGTTGCCCACGCAGGAGTGCAGTGGCATGCTCTCAGCTCACTGCAGCCCTGACTGCCTAGGCTCCAGCAATCTTCTTACGTCAGCCTCCAGAGTAGCTGGGACCGCAGGCACTTGCCACCACGCCCCACTAAAAATTTTTTAAATTGTTGCCTTTCTTGAAGTGTTCTCTGCCTGTCTTTGTCACAAAATTTCATTTTTCTCATAGTTAATTTCATCTCTCCGGTAAGATTTTATTGGTGTTTCTTTTATAACTTTGCAGTTCTTACACCGTTTGGTGATTTTCATGTTTCTTAGAAACTTTAAACCTTTAACTTCAAACATTAAAATACAAGTCTTTTAAGTACATGAGTGCTTAGAAATGTACATAATGTTTATATACACTTATGCCTTACATTAAAGTCCAATATGAGAAATACATGTTTAACATTCAATAATAATTTTAAAAATTTGAGAAATAAACTCTCATAAATGCACACATTTTTATAAACTTGCTACTTGTCTGCTGTT GATTTTAACATTTSEQ ID NO: 25 GCAGAAGCTTTTCCTCTCCTGCCCTGACGGCGAGTTTTCTCCATAACTTGCTCCCGGGGTTCCCCTGCGGGTGTCCTCTCCCATCCAGACTGGCTGGCCATTCCCGGGACTAGACGGTGCTTAGGGCGATCGGGAGCTCTGATCCGGCCTGATTTTTCTGATCATCAGCTTCCTTTCTTTTTCATACTCTGATTCTAAAGATAAAGATAGAGCCAGTTTCTCCCCCTTTGGTGTTTCAAATTCCTTTCTCATGACTCTTCAGGCAGTACAGTGAAGAGATTAAGTGCACGACGTGGAATTCCGGCCTGTCTCCTTAATAGCCTTACGATCTACCTCACGGAAGTGTTATGAGGGTGGAATGAGTTAGTGCACATAAAGCCTGTAGCATGGTGCTTGGCCCAGAATAGGCCCTCAGCGAATGTTAGTTATTATGGTGATGATTAATACTTCAGTTTCTCTGTGCTCAGCAAACACCACCTCCCTGTGTGGGCACTGCATAATCCCCTCGCCACATATATTCAAGGTCCTACCCCTGTCTGCTTATGCTATGGTCTCTAAGGAGGAGCAATGGGGGATGCCTGTCACCTT CCAAGACTTTTATSEQ ID NO: 26 TTGTGTTATTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCCGGTCTCGAACTCCTGACCTCAGGTGATCCACCCATCTTGGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACCATGCCCAGCCACATAGAAAGATTTTGATTAGATATTAGGAAGGATTTGGGCTACAACAAAAACTTGTTGAACGATTAAGTCAAAGCATACTCCTTTGAATACTGAAAATGTTTGCTGAGGTTGTCTCAGTTTTGAAGGACTGCTTTCCATTCCATTTTTCTTGTTATCTTCTGATCTAAACCGGTGATAAATTTTATGCTGTCCGATGACAGGCAGGAATGGTAACAGAACTTGCAGATAATAGGTTGCTTACCAAAAGTTATAGATTGACCAAATGAATTTGTTTTCTAAGAAAATGGAGGGCATTAATATAAAACTCAAATTCCTTGGAGTTACTCAAAGCCCTCAAGTATATTTGTCCTTGTCCATTGCCATGGATGTTTCCAAAATGTAAGCTTTGATGAGAAGTGGTAAAGAAA ATAAGGCATTCGSEQ ID NO: 27 CCTTAGTTTTTTGCTGTAACCCAGCTCATGGCCAAGGCATCGTTGCATAGATTTGAGACACTAGAAATCAAACCTGAACTGCCGGAAGTGCAGATGCCGCAGCAGACCTCCTGGTCAGGCTTCAAATCCCACCTTCCACACCTGTTCCTGGGAGATGAAGTTTGTACCCATGGTCATCTGTCCCTCTGCCACTCCATTCAGATGTGTCCACGTCTCCATGGCCCCTCCATCCAAGAGAAAGCAACACACATGTGCAACCAGGAAACATACAAAAGTGCCTTGCAGCCTTGTGGATCCTTACCGGCCAGATAGCCATGGGCTGTGGAATGGAACATAAATATCACACAATGCAGTTTTCCTTGGCAGCAAAAATGAGCAAACAAATAACATACATAATAAAATGAATAATTTCACAGGCACAATGAAGAGTGAACAAAACCAGGCACAAATGAGAACACCCTGCGTGATTCTATTTATACAAAGTTCAAGAATACGCAAAATGAACCTGTTGTAATGGAAGGCAAAACAGTGTGATCTCCCAATGAGATACCATCTCACACCAGTTAGAATGGCAATCATTAAAAAGTC AGGAAACAACAGGSEQ ID NO: 28 TTTTGCCTAGTTTTTCTGTAACCTGCATAGTTGTTTAAAGCTATAGATTTTTAAAAAAATGTGTGCATTCAAGTTTGTTTAGAGTACAAATTAGAAAAGAAGGGTGCTTTCCTTTCCCAGATAAAACCTTTGCGGATGTCTTGGGGGTTGATACTGACCTCTAGTGAAACCAGTCTAGGTCAGTGCTGTAGCTGGAAATTTAAAATCCCACTCAATTGATTGGAATCAGTGCTAAGAGTAAATTAAGCTAATAACAACTCTCTTTCAATACAGGGAAAAGAAACAGAAAATTCTGGACATCCGGAAAAATGTTAAAGATGCTATCGTGGTAAGGACTTTTTTAAATGATTGTTTACTAGAAAGGTCAAGTGCTCTTCATTCTAAAACTGTGTAGACAGAAATTACATATTGTAGATTATCATACATGGAGCCTAAATGTTTATCCATATTTTTTTTCTTATTCCATTTTAGACAATTGTTTCAGCAATGAGTACTATAATACCTCCAGTTCCGCTGGCCAACCCTGAAAACCAATTTCGATCAGACTACATCAAGAGCATAGCCCCTATCACTGACTTTGAATATTCCCAGGTAAGAAATGAAACAACAGACATGAGACTCTTGAGTGGCAGGAAAT TTTCCATCTCACTTIn some embodiments, the methods disclosed herein utilize at least 15,at least 20, at least 30, at least 40, at least 50 or at least 60nucleotides of these nucleic acid sequences.

In some embodiments, the methods utilize one or more of SEQ ID NO:19-28. In additional embodiments, the methods utilize all of SEQ ID NO:19-28 to detect an aneuploidy of chromosome 18.

The following nucleic acid sequences are less methylated in fetal DNAthan maternal DNA, and can be used to detect fetal aneuploidy ofchromosome 21. In some embodiments, the methods disclosed herein utilizeat least 15, at least 20, at least 30, at least 40, at least 50 or atleast 60 nucleotides or all nucleotides of at least one of the followingnucleic acid sequences:

SEQ ID NO: 29 AACCATAAAAGGGGCAGAGATAGACTCTGTGGGTTGTCTTTGGAATATCCCAAGGGATTTGCTCCTCTCTGATCATGTAGGTCTGCGGCTCCAAGGGCAGTCCGTCACCACCCTATCTGGAGCACTCTTCTATCCTCCTCCCATCTCTGTCCTCTGGCGATGACCACAGCCCCGGGCACTGCTAGTTGTGCCCCTTGCTAAGCTCCCATATTTTGTGTTAAGGAGCAAGGATTTTCACAGGCTCATAGAGCATTTGCTTCCTTTCTAGGGTAATTAAGACTACACTAGAAACCATCTTGTCCGGTTCTCTTTTACAGATGTTGACATTAGGGCCCCAAGAAGATACGTAGCCTGCTGGTGTCACTTAGGGAATTAATTCCTGAGCAGGTGTCTGGTGCCTGGGGCCCTGCTCAGACCCCATTCTCTCCTCTGGTTCTCGATGGCCCTCACCTCCCATTTCTTTCCATTTCCCAGAGGAGTCTGGTTGAACTTATGGCTTTTTTTGCCTTGATTAATTGACTATGAATTTATGTCCCCTAAAAAAGGATTAAGTTGCATCATTTCTTCTTAGCATGCAGTGCTCAGAGA CAACCCAGCAATTSEQ ID NO: 30 GATAAAGAAAGTAATTTTATAAGAGAGGAAATAAAATGTAATGCTTTTTCCAGCATAAGGTGAATGAGATCATCTGGGTTAGGAAGTTTTTCTATGCTGGGAAGATATGATAAAGTAATTGAACATAAGTGCCTAGAATTTTATAGGAAACTTCCAAAGTCACTTGATTTAGAAGGACAGATGAGGCTCAATATGGCTTCTTCCCTCAGAAGTGGCATAGGTTACAGCAGTAGAAACTTAATCCAGCCTCAGAAAGAGTTCCCAAAGCACTCAAAAGTTGTATGCAAAAGACTTAGAAAACCGGGCTGGAAATATTTTATACAGTAACCCTGAATGCTTTAGGATGGTTACTTTTTAATGTATTCCATGCTTGTCTAACACCTGCCCATTCCAGGGGGTACATACGCAGGGGCCTCTCCAGGCGAGCATCTCAGTTATACTTCCATCAGGTCCTGGCAGGAACACTGGATAGGGAGGCTAGATTCACTGCACATCGCCTCTACCATAGCTGCTCAGACCTTCATGTGCACACAAAACACCCAGAGATCTTGTTGGAACACAAATTGCCAGGGTACAACCCAGGGGAAT TTGCATTTCTCACSEQ ID NO: 31 AAAAATTTTAAAAAGAAAAGTATGATGATGATAAAATGAACCACTAAAACTAAGAACAATAAGTGAAAAAAAATCACTTTTTTCTGAGTGGGAGAAACAAAGGTATAGAGACGGTCTTGCTAAAGGAAAATTGTAAATTTAAGTAGCAATAAAGAACGTGGCATAATTTTCTCCAGCAGTATTCAGTGGCAGAGGAAAAATATATGCATGCAAGTAGTTTGGTTGATTGAATCTGTGGACAGAAAAAAATAAAAAAGAAAACCAATTGATGAATTGTGGGAAACTATTTCAAGCAATGGCCCGGGTCTTCTGGAGGTATTAGGGAATATTACAGCATGAAATGACATATCTTAATAAAGAAGTTTAGAGAGAATAAGAAAACAGTATAGTCGAATATTGTTATAGTAACTACAATAATTAACTACAACAATTCATCATTATATTCAAAGACAGAGACCAAAAGATCATATTGAAACCAACCCAATAGTCTCATGGACAGTTGTTTTTGGATAAACATAGAAATTGCCCCTTTCTGGTCTTAAAACTTGAAGCCGGCCAGGCGCGGTGGCTCACGCCTGTAATCCCAGC ACTTTGGGAGGCCSEQ ID NO: 32 TGGATTTAAAAAATATATATATCACGAGCCGAAGTATGAACCACGAGAGATTATCTCCACCCCCTGACTAAAATCTCCATTAATTAAACATGAATGCAAAAGTAGCCTCTGCCTACAATTCATGTGTGCCATAAAGATGAATAAATAATCCTTACTGAGTGCTTTGAATCTCTGAAAAGAGAGCGAGCATACAATTGTTAGACATCTTTACACCTGCCCTCATGTTATCTAATAAGCCCATTATGGGAGTTTAATACAAAAATATATAACAAAATTGTAGAACGTAGAAATATATAAAATCCGGGATGAGCCAGAATTTACCTTGTAACTAGAATTACTTCATTTATAATCTATAATGCTACAGATGTATATTCTCAATAGTATCAAATAGGTTTATTTTTTCCAACCCTTGGATTTCTTCTTGCATCTTCACCATCTCTAAGCAAAGTGATTCCATAGCTAAAAGGCAGAAATGTGAATTCTTCTTCAAACTTGTTAATACATGTTGAACAAGATGCTGATAAACCCAATCTCTAAAAATATGAAATATCTGATATTTTGTAACTATAAAAAATCTTAAAAAGGGAA ACAAATCCAAACTSEQ ID NO: 33 ACTAGAAAGGAAAGAAACTGCCCTTCCTTTAAGGAATGTGGAATCTTTCCTTTTGAAGATCAAGATTTGGAGCCCATAATCCTGTGGATGGATGACGCCAACATTCAGCTCTGCTGCTTATTCCCCATAATTTCTCCGCTTGACCTGTGACGAATCAGCTTCTGCCTTTCCAGGGACTTAGAACCTTTCTTATGGGACTCCTCTCACTCTGAATGATGGTGATTAATTACCAGTGTACTTTAAAAGTCTCCTCTATTTAATTACATCCTCCTGAGTATTTGGACTAATTCCTCTTAGTTTCCGGTGCAGATCGGAATGTTGCCTTGAGCAAGTCTCATGTTTCTTTCTCCCGCTTACGCAGTCAAGCACATACATAGATACGTAATAAATATTGATTAAGGTTCTGTATTAGGCTGTGTTTTTATCAGTAAGGATTAATAGGCATAAATGAGAGACATGATCCCTACCCTGGAGCCTAAATGGCATGTGTTATAAAGACGCTTTCAGGCTGACCAGTTCTTCTAGTGCAGCTGAGAAGACAGCCATGCCTCCTCATTAGCCCACTTTATAGGGAAGAAATGGAGACTC AAAGTATCTAAATSEQ ID NO: 34 TTTTAGCATCATCTGGTGAGGCTAAGGATTTTCAGAATCATCAGATACTGGTTCTTTTTTGTTCGATGTTCTTCCCTCAATTTATCTCTTTCCTCTCCCGCTTCACTATAAATAACATGAAGAAACCAGGTGATACACTTAACACTTTGTTTGGAAATAGCCTTAGCTATGTGACCAACATCATCGCTTACAAGTCCTGCTCTCCATGGACAATTTCATTAAACTCTCTGGCACACATGGGAAGGATCCTCCTTGCTCCGATTTCCTGGAACACCTGTCACATTCCTTTCTGAGTTCTCCCCGGAGGATTCACCTTTAGCATCCCTATTTCCTCTAGCAGTCTGTTCATGGTGATTTGGGCTTTCTCTGCTGTGCTACTCAAAAGTCTCTTAGCCTCTACTCGCTGCCAAGTCCCAAAGTTGTTTCCACATTTTAAGTTCAGAAGGTTTCCAACTTACAATGGTTTGGCTTAGGATTTCATAATGTTACGAAAGTGATATGGGTTCAATAGAAACTGTACTTCAAGTGCCCATACAATCATTCTGTGCTACATTTTCAGTACAACATTCAACGAATTACATGAGATAC TCAACACTTGATTSEQ ID NO: 35 AATAAAATAGAAGGAACTTTGATATATGAATCAATATGGATAATCCTTACTCTTATCGAAATCCTTACTCTGAGTCAAAAAATCCAGGCAAGAATGCATACACAGTATATCCCATTTATATAAGTTTTTTAAAATGCAAAATACTTGGATGAAAGGTGGTCAGTAACTAAGTTGTTGCCTGAAGATGGGCATAGAGGGAGCCTTGGATTAAGCAAGACAGTAGGACACTTTGGGTGGTAATGAAAATGTCGGTTTTCTTGATTATAATGATGGCTTCAAGCACAGATACAGATATTAAAACCGGTAAATTTTTCAAATATGTGCAGTTTATTTTAATTAAATTATTCATCAATAGAGTTTAAGACACAAACAACTGGATTCCACCAGAAAACTAAATATATTACCTTGTTAAATTGATGCCTAAGTGAAGCATAGTGCCTCATAGCAGAAAGGGTTAAATTTATTTCATTAATCCTCTGTTTTGAAAAAGTGATCACTAGATAAGTTTGTTCAAAAAGAGACAAAAATAATAACAAAAACAACTAGGCTTTGACTGAAAATCCAGTGCCTTAAAAACTGTAGAGAATT TCTACACTGTTTCSEQ ID NO: 36 ACACCAGCATGGCACATGCATACATATATAACTAACCTACACATTGTGCACATGTACCCTAAAACTTAAAGTATAATAATAATAAAAAAAAGAATGCTTGCTTACAAATTGTGTTTCTTTAGCACTTAATGTTGTTTTGTGTTTTTCAAAAAAACAGCATCCAAATATACTGGTTGACAATTTGTTTCCAGTTTTTTTCTATAAATGACAAATAGGATATTTAATTTTTGTCTAGCGTAGAGAAGAAACATTATAATTTTGACAAAAATATGGTTTCACCTTTCATATAAAATTCTAAACCCGGTAGCACACTTACCTGAAAGGTGAGAAATATGACATTGCCCCAGTAAGTGACAACATCTGTGGTGCTATCACAAGGCTAAATTCCTTTATTGTGTCTTTTATAAGATACTGTTAAGTCTAGCAAAATACCATTTTGAGATGCTACTTAAACGTGGCTCAGAATAAATAAAAAATTGTCAAAGAAACGTTTTAAATTAACACTTCTGATTTTAAAATATAAAGCAATCAAAGACAGTGGTTTTCCAACGTGAACACACCGGGTATCAGAGGAGTATTATTGAGGCA ATGTAAAGAGTATSEQ ID NO: 37 AGGCAAGTAGGCGTTGGATGGCAATCCTCTCCCATGTAACCGTGCTACCCCATGGTATGTCAATAATGAAAACAAGTTATATAACAGATTAAGATTGTCTAAAATGTATTAACAAGAAGCAAAAATAGTCACATCAACTGCTCTTGTAAGCTTAGAATACTAAGGCTGTTCAGACATTCTGAAAGCAGGATAAGCCAACCTTCTAGTTTGGGTCAAGCAACATTGTCATCTCTGAAATGAAAGTGTGTGGCCAAATGGAGATGTGAACCTACCATGCTGCTCCAGAGATATTAGTTTCTGCCGGATTTGCATATCTTTACATCGAGTTCATACATGGGTGTTGGTGCCTGTACACTCATTCTATCCAGTCATGTAACTCAAGCACAGCTCATTTTACTAATGTTTGTATGCTAATTAAAAACTCTTAGGTATAGATTCCTATAATAATATTTAAAGTTATTATGTAGCTACTTCTGAAGTGTGAATACCTGAGTAGGGTACTTCAGATGTTCAATACTCCCTTAGAACAGTGAATATAGTAAGCACTCAAATGTTACTTACTACTTTTCTTGTACTTATCTTGGTTGT CATCTTCATTAGTSEQ ID NO: 38 GCCTCATCAAAGCATTGTAAGAACTGATAACCATCTTCTAGAAGTATCATAGTGATATTAAGAACACACATCACAGATCATAGTAAATGGCTTTAATTTTTTAGCGAAATCTCACTACTGCAAATGCATTGTTGTCCTAGCTAATGAATGCATAGAGTATTGCCTGCAAAATAATAATTGAGATTCTATTTTTAAGAAGCTTAGAACAGTACATGGTGCATAGCAAAGACTCTGTGTATGTGAAGCCAGATTTTAAAATATGGTAACAAGTGTCTGAAAATATGTGGCTCAATTTGTCTCCCGGTTACTTTTCCCTCTCCCCCTTTAAAATGTAGAGGAAGGAGAAGAAGAGATAAGAGGTTTGTGAGTGAAGACAAGGGCCCTTTAAGGCCTGGGAAGACTAACGCCATAGGGATCTCCCTCTGCCTTAAAAGGCACAGGAATCTTAGTGGGGAAAAAGAAGTGGTGATAAATAGCCAGTCCGTGTGCCTGGAATATCAAAGTCAGTGCGTGCCAGGGATCACACTGCGGGTCACGTGCACTCTGGGTCTCTCTCTGCAAACCTGCCCTGCCTCAGTCTGGGAATAT GCAACTGCCTAAGIn some embodiments, the methods disclosed herein utilize at least 15,at least 20, at least 30, at least 40, at least 50 or at least 60nucleotides of these nucleic acid sequences.

The following nucleic acid sequences are more methylated in fetal DNAthan maternal DNA, and can be used to detect fetal aneuploidy ofchromosome 13. In some embodiments, the methods disclosed herein utilizeat least 15, at least 20, at least 30, at least 40, at least 50 or atleast 60 nucleotides or all nucleotides of at least one of the followingnucleic acid sequences:

SEQ ID NO: 39 ACCCACCTGGTCGCATCCTGCGTTCACCAGTTCCTGCAGCATTTGCCGGTTCACTTCTGCAGCTGCAGAGGTGCCCGATTCATTAGCAAAAGGCAACAAGGAATATCTGATTTCCCTCAAGGCTTTCTGATAAGGTCCGAACTTTGGGGTGGCTCTCATCTGCTGCTGCTGCCTGGTGGCATCTTTGCTCCCCAGGACTTTGGCATCCAGGGAAGTGTCACTGTTTGGTCCTGCGGGTAGCCCCTGAACCGAAGACTTGGATGGCTGTTTTAACCCCTCACGAATCTCTTGCAGTCGCTGCCGGCTATTTCCAGAATAAGTCGTGGCAGGAAAAGTCTTTGGCCTCATTGTTAGTCCAGTTTCCTTTTACCATAAATACAATCTTCTTAAAGTGTTTTATTATTTAAAAAAAAAAACTGTCAATAGTATCAGTTTGTTGTAAACATACTTTCAAAATAATTTCCTTTTGAAAATGTTCTTTCCTTCCATTTTTGTAGTTCCTATAGAGAACCTAAAATTTTCCAAAGTCTTCATTTTGAAGATTATCACTCTCTGAAAAAGAAAATAAATGGGAGAGAAATGTTCATT TGTCATTTTCACGSEQ ID NO: 40 GGGTGCCCGTAGTCGCCGCTGACCTTCTCGTAGTGAGGGCAGAAGACGCTGTCCGCAGTCCTTAGCGGGATGATAATGTCACTGGGCTCTGAGCCGTTGTTGTTGCCGCTGCGCTTGGGTGTGGCCAGTGTGCTGAGCGACAGCGTGGTCGTGTGCTGCGGCGAGTGCTTCCTGTGTCTCCTCCGGTACTTCAGCAAGAGGACCACCAGCGTGATGATGATGACGATGAAGATGATGCATCCTGAAGCAATCCCTGCAAATAAGGCCACTTCGGAACCGAGGATGTTGTTCCCCGAATGTCCGGCGCTGTTGCCGTCTGTGCTAGAACCTGCAGACGCGGAGACAGAAAAGGTCAGAGATAGTTACTGCTGTCCCAGTGCAGACGGACTGCTTTTATGACCCTTAAAAATGTGAAAAATAAGCCAGGCTTATTACTAACTAGAAGCTGGACTTTGCCTCGCCAATACCTCGTCTAAGAGCTCAACGCAAAAGGAAATGAGAGGTATCTCTAGGAGGGACTTCCCTACCCTCCCAAGCCACAGGGTTTCCTTTGTATCATTATCCAGCAAGAGCACAGACTGTCGGTGA CAGCCAGCCCTTGSEQ ID NO: 41 TTTTTTACGTAATGCTCAAATAGACCTGGGAGATAAGCACTGCTATGAATCCCCAATCCCCATGTTACAGACAAAGAAATTAGGGCCAACGAGTGGTACAACTGGATTAAAACCTAGGCTTCTGGCCCCAGAGCCGCACACTAAACTGCTGTGCTTGAGCGCTATAAAATAACCACATTTATTTATGTATTTTCTCTAGTGATACTTATTTGCAATTTTCCATATCATGAACAAATTTCAGTGAACTTCCTTATGCCTACCTCTTGTGTGTATGTGGACATTTCCTGGGGATATATACCTCCGGTGGCATACCTGTGAAATCCTTTGCTTATTTTTTGAGAGAACAGAGCAGAGCAGATGCTAAAATTGCCTTTTTTCCTTGAGAATTTTTTTTCTCTGATAGAATACATGTTCCATGAAGGCAGGGATTTTTGTCTCCTTTGTCCACTGCTCCATTCTGAGCATCTAGAAGAATGCCAGGCACATGGGAGATGCTCAGTAAATATCTGTGAATGTATTAATGGCTCCTGAATACGACGCTCTTTTAGCCTAATTATGCGAACTCTTTTTTTTTTGTTCCCCGCCCCA CCCCCCTCCAGACSEQ ID NO: 42 ATATAAATCTTCTTTAATAAAACAATTCTGTGGGACAGATCTGCTACCAAGGCTCAGAGAAGTTGAGTGGCCTGACCACAGTCACACAGCTGATAGGTAGCAGGATTGGGACTAGAGCCTGGGGCTCCTGCGCCCCAGGTCAGTTACTAGGCATGAATAGAAACACCGCACTGCAACACTGAATGTTAGCCGACATGGCAGCTAAAATGCACACATTAGCATTACCAAATGAGTGAGCCGATTGAGCAAGTGAGTTCTCCTACCACTACCTCCCCTTAGCATTGGCTTTGAAGACGGTAACCGGGAAAACTCAGACTGGGTCGCCCCATGTTATTGCGCCAGGACTGGAGACTCTGAGAAGCTGGTTGCAACATTTGGCGTCTTCGCTTCGGATGCTCTATAAGCCTTTTGAGAGATGAGCCTGAGTTTGTGGCGGCAGGCTCCCTTTTGAAAGAGCCACCCAATCTGGCCTCCCCAGACATCTTCCTGGAGGAGGATTGAGCATTATTCCAAAAAAGAAGGGTGGCCTCGAGCTGGCCCATCAGGCCCTAGCAATACCCAGCTCCTCATCAGGATGGGTGCAATGTT TGTTTTGGACTTTSEQ ID NO: 43 ATATATTGCCACCTGAAGAGTTGGCTTTCAGAGCTCTTTGGAATTTGGAATTGTAGATGGAGGGTCATGGATGTGTACCAAAAATGTTCTTAACAGCCAAGAAGTCCACAGACTAGGCATTGTGCAACAGTCAAAAGCAACTCTAGACAGCTCCCCAGACCAGCACAGTGCTGCTTGTTCATCCTTGCTCCTAGGAAGCAGGAAGTACGTGATATCAGCTCTCTTGGAAATAACTGTCCCTTTAAGGAGTAATTGTCTTGAGTAACCCCTAAGGAAGTATTAAGTATTAAAATTCTAGGGCCGGATTCACCATCCCCTAAGGATTCTGCATTTCTCTCTGAAAATCTTAAGTTCAGAATAACCAACCAGTTCCTTCTAAACATCAACAACCTTATGTTAAGTTCTCTGACCCTTCCCTGGTAATTCGTAAGTTTATAAAATCTCTCCCCTTAACTTAAGCCTTAGTTTCCCAGATGCCATCACTTTGAAAATCTGCCTCAGAAACATCAGTACCTCCTAATGGGATGTGATAGAACACCCGAAAAACTGCTGAATGGTGTGGTGCTTCCCCAGAGCCCAGGGGACAGT GTCTGGGGTCCCASEQ ID NO: 44 GCAGATGGTATGCCAGGGACAATATACAAGACAGAATAGAGTTCATTCAAGTATTCTATGTTTTGTTATTTAGTTTTCAAATATGAACCCTGGATGGAACCAGGCTTTGTTCCCCCAAGTTATCAGCTAAAGAGAAAGAGCTCAGCCAAGTGACAAGGTGTTGCCGCAGTGATAACTCAATTTTGTGGTTTTCAGAAATGCAGAAGAAAGAAGGTTGGAAAGAATGGGCTGGCACTCCCCGAAGAGTGGGGCTCAAAGTTCTGGTCTCAGCAGATCTTTGAGGGAGACGTAGGGTCTAGGCCGGCAAAACCAGAGAGAAGGATAGGCTTTTAGATGCTCATCCCACCCAAAACCATGAAATAATCTCCCCTGTCTCCTGATTAGTATTTGTCAGCCTCATTTACCCTTAAGATTTGTGCAAATGAAAAACGTTAAATGCAGTTCCTGGATCAGTAATTAAACATAATTATTTCAGTGGAAGAGATTTTTGTTTTCATAATCTCCTCTTTGTCTGCATAGAGCCAGCTCTTGGTTACATCTGCCACTGATAGCCTTTGCTCTGTTTACTTTAAAGAATAAGCCACTGTT TGATTTGGTTTCTSEQ ID NO: 45 TATTCGCATGTGTTTTTGTGAATCATTCTGTCGAACAAGATAGAAAAAGAAGGGCCAGTTCCTAGGTTGCACTGCGTGTCTGTGTGATGGAATGACCCCTGTAACTCCTTAGGCCTTGATGCTTCCTGTGTTGTGTGGGATACACAGAATCCACTTGCCTTCCTCCCAGGGCTGCAGAGATGAAAAACTATGACACACATTACAATGCTCTGTAAACTATGTTTATTCTACAAATGTGCATTATGTTATGAAAGTGGAGTAACTAATAACTCCACACTTCTGCCCTCAGAAAACTTCCAACCGGATCATCTACCTCTTTTTGAACGTGCCCAGCAATGGAAGGTTCCTGCACGGCCTCATGTGTTGTTGCATAACCCTCAACATTAGGAAAGGGTTATTTTTATCCACTCAAACTTTCATATGTTCCAGTTTAAGTAATTTACCCTTTCGCTGTGCTCAGAGGGGACAGGGACTGAGGACACGTGGCCAGATCTAATTGCTTCCTCCCTTCCAGGATGAAGGATCAAAATTCTTTAACCTTTCTCAATTTCCAGTGTTTTAGGCCTATTTTTACTTGATATATTCCTT CTTCAAGGGACAGSEQ ID NO: 46 AAGAAGCTGGAAGCTGATTTAGTAACTTTATTGTAGTTTGCTTGGAAGGTAAATGTATTATTGTATCCTTCTGTTCACTTAAACTGCTGACCTAGAGTAGGTTTGAATAACACTTTGTTTAGTTTGGCCACAAAATGCCTGTGAAAGGGGAAATTCTTTGGGCAGCCAGGGCAATATCACTTCCTTATCTCCCAGTACCCAAACACCTTTAGTTTCTCCTTCCTTCCTTCCAGTTGCTTTCAAAAAGTGGCGATGAACTGCTTGTATAGTTGCCATTTAGTATCCAAGGGGGATTGATTCCCGGACGCCCACAGATAGCAAAATTCCTGGGTGCTCAAGTCCCTGATATAGAATGACATAGTATTTGCATAGATCTTATACACATCCTCCTGTATACTTTAAATTATCTCTAGATTACTTATAATACCTAATGCAGTATAAGTGCTTTGTGAATAGTTGTTATAATGTATTTTTAATTTGTATTACTTCTCATTTGTTGTATTGTCATTTTTTTCCCCTGAATATTTCCTACCCATGGTTGGTCGAATCCTTGAATACAGAACCCAGGGATACAGAAGGCTGACTATG TGACATTTCTTCCSEQ ID NO: 47 AGAGCTTACCAGTGTTATTTTCCTTATTACTTCTGAGTCTGGCATGCTAGTTTGGTTAGTGATATATTATTATTTAGGCCAAAGTCAGATATTTTATAACTACAGAAGCCAGTTAGTTACCTTCCCTTTGTTTCACAACCGAATACTATACTATGCACCTATGGTCCCATCTAAAAGATATTACTGGTCATAAGAATGTGGCTGTCTCCGAAAAAATTGTCACTACTAGAAAAACAACTGAAAGAACATAGTGTAATTAGAACAATATGGCCAAACACGTTTCGTTAATACTGTGTGTTTCCGGAGGAAAGGGAAATTAGCAAATATTTTCCTCCTTTTCAACATGGCTATGTTCCTAAGCAAGATAGCCTTATCCTGCTGAGGAGACAGAGGATGTGGCATAAGACATACAGAAATTGACAAACATATTAACCTTGTATAACTAGGACTTTTTTACAAGGTATATTTCCCTTGTAAAACTAGGACTTTAATTTTGAGAATAGGATGGGACAGAGTCAAGTGAAAGAAGCCTTAGGTATCACATGGCACCAAGGAAAAACATCAAAGATTTCTGTCTGCTGCCTTTTC CACATAGTCCTTTSEQ ID NO: 48 CAGTAAAGAGACCAGCCACAGATAAGTAATTGGCAACCAGCAAGGTAATAAATCTAGTGTGGGGGACTGGCTGACGTTTGGTCAATGTCTCCTTCCGAAGCTCCACCCAAATTTCTCCTTTTTGCTTCTCAGTCACAGTATTTCCCTCCTGTTAAATAGCATTAGTCTGGGGAATAAATGCTCTTCCCTGTCTACTACACTGTGGGAAACGTCAGCCACAGCCTCCAGTTTGTTAGGTCAAAAACCTTGAAGTCATCCTCTCTTCCTGTCGTAGCCACGTTGATCCATTCACCAGAAAATCCGGCTGGGCCTACCCAAATATGTAGAGAGCCCAGCACGTTTTTACCTCTTCCGTCGCCATTGCCTTGGACCAATGCCTCTCACCTGGACTATAGGAAGGTCCTCCAGATGGCAGCCCTGACTCTTTCCTGCCCCTCTCCAGCCGCACTCAGCCTCACAGTATCGATGATGATTTTCTTCACAGTCAAGGAACAGAGCTCCTGGGCTGGAACCTCACACCCAGGAATCAGGGTCTGAGCTGAGGTCCTGACAGTCACCTACAGGATTCTGAGCCTTACCCCATTGCAC TAACCTTTCGAAAIn some embodiments, the methods disclosed herein utilize at least 15,at least 20, at least 30, at least 40, at least 50 or at least 60nucleotides of these nucleic acid sequences.

The following nucleic acid sequences are more methylated in fetal DNAthan maternal DNA, and can be used to detect fetal aneuploidy ofchromosome 18:

SEQ ID NO: 49 TGTATAGATGCAGAGTTTTACTACTGAAAGGAGGAATACTACTGTTGATTTGTTTTTGTTTCTCGGTTGTTGTGCGTGTGTTTTGTTGTTGTTGTTGTTGTTTTACCATCTTGGTCCTAAGTAGCTCGTTTGCCGGCCCAGCCTTAATGGCCAGTTGGCTCCAAGTCAGAAGACATGATCTCCTCCCCCATTCTCCATGCCATTGTTTAAAGCCCCTCCTGAGGAATGGGCTGCCTTGGTGTTTTGTCAGTTCAAACCACATCCTGCCTGTTTCCACTTTCCATAAGACAACTCGCAACACCGGTGGTTTTCAGATGTGGCCGGCTTCTTGGTGAAGCGATAGCAGAGGCCTTGTTCACAGAAGTGAAAATAATTCACCCAGTGGTTAGCACATCAGGTGTGGGCATTGAGTGTACCCCGCTCCCTGCTTGATCCCAATCCCTGGTTGGGTTTGGGAGTGGACGGCTGCCCAACCTCCTGGCACTGTCTTGACCCACAGCCTTCTCTGGGATGAGGACTAAGCCAGAAGCAGTAAGGACAGAGGTGTCTCAGGCTGTCCAGGCCTGGCCTGAATCCCATGACAGCAAG GGTGTGGCCTGCASEQ ID NO: 50 GCTGAGGGATTTGGTCATATCATTAGCCCAGACATAGCCTGACAAGACCAGCCCTTGTGTAATGAGATTGGTTTGAGGTAGGAACTGTACTTGCTTTCTATGATGGGCAAGAGGAGACCCCAGGCTCTGGATGGGAAGCTGAAGGCTGACCAAGGGTGGATGAGGCAGAGGGCCTGCGAAGGGGACTCCACAGAGGCCACACGCAGGACAAGAGCAGCACTCTACTCCAGGCTTCATCCATCGTATGGAGGTCTGTGGTCATTTCACCCATTCATCTTGCTCCTCTGGAGAACCCAAACCCCGGAAACCACAAAGGGAATCGACTGCTGCTTCACAGGAAGGGTTTCATTCATGATCACATTTCTTTACTGAAAAAGATGGGTATATTGAAGCGTTGACTGCCTCCTCAAGAAGAAACAGGAAGTGATCATTACCAAACAGAGGCTCCCTTTAAATTTAAAATATGTAGCCAAGAAAGAGGCTAACACTACCTAAGAAAGACCCCAATTAAATGTTTAAGCTAAAAAAAGCAGAGGGTTGGGGATAGGGCATGTTTTTGTTTTGTCTTCCAATTTCAGTGTTTTTAAA GGCTGAGCACCAASEQ ID NO: 51 GATTTTTGCAGAGTTATTTTTAAGCAGTACAAAGTAGAAACACACTTCTTGAAACAAAAATGCTTTGTCATTTCTTAATCTCTACCTTTAAAAATAGCAAGCAGCTATTTCACTCTGAGGAGCGCTTACACAGCCATCACTGCCCGCAACCCTGGGACCTGAGGGGGAGATGGGTGAGTCTGGAGAAAAGGCTGTTTCCTAAGAGTCTCGTTGCTAGTCCTGCTCGGTCACATGACCTCACCTGTGGTCCCGAGCGATAGTGGGCAGCACGCAGTTTTATTACATGAGGCAGTATGTCTCCCGGTTCACGCTTATTATAGCAATAATTCCAGAGCTTCTCACTCTTGGTTAAAACAGAACAGAAACACAAATGCAATACCAAGCACAAAACCACGAACATCTTTGAGTAACTGGGCTCTTCCTCCTACACAAAACGTCCTGACGGGTCCCTCAGAGTGAATCTATGGTCACGACCCGGACCCCGGAGGCCTGTGCATTGAGTATGCGCTAAGGTTTTTAATACAACTGGCTTCTTTCCATTGTCTCCCAAATATCCCACCCCAGGTAGTTGGGTACTGAACACAAACT GGTCATTTTGATASEQ ID NO: 52 CATGGTGAGACCCCATCTCTACTAAAAATACAAAAAATTAGCCAGGCATGGTGGCGGGCACCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGTGGAGCTTGCAGTGAGCCAAGATCACGCCACTGCACTCCAGCCTGGGCAACGGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAGGGGAAGTCACCAAGAAAGGGATAGAGGTCAGTGACATGGTCATCACACAACTCGGTCATTTAATATGGCAAAACACAATACCAAGAACTGTGCATTAGCCCGGAAGGCAAGAGGGTTGGTTCTGCCCCAGCTCTGCTGCTAATGAGCCCCTACCAGGTTGAGCAAGTCTTTCCCCACTCTCCACCTCCATTTCTTAATCAGTTTCATGACAGGGAAGGATTTGAAAGTCCTTTCCACCTCAGAAGTGTTGATTATTCTCTCTCTCCTATAATCTGATGAAATCAGCATGACAACAAGTAATTTAAGTAAAGGACAGAAGGGTTACCCAAGAAGAGGCAGGAAAATGTTAACGTTCTATAAAGTTTTATTCTTGAGTCATAGGAAAAT GCTCTCCAGACGTSEQ ID NO: 53 CTTGTGTTACAGATGGAAGATAAGATTGGAGAGGAGGAAGAGTTAGTTTTGAGTCTCTGACAACAGGGCTGCCACTGCCAGATTGCATCAAACCGTTTTAGGTTTTGTCTTCTGGGAAGCACCATTGCATCATAGTCAAACACGAGGGCCAGGAAGCCCAAGAAGTGTGGGTTTCAATTCCATCTCCACTTATCAGCCACACTTTTGTGCCTCAGTTTTCTTATACACAAGATAAGGCTGGTAATCTGGCCAAACTTTCAGGATAAGAATTATAGACATTTAAGAAACCCAGGAAACTGTCCGGCACATAGGAAGCATTGATATTACTGAGTGATAGCACTGAGCCAGTAAAACCTAGACAGAAGTTGGAAGATAGGGCACAGTGTTCTTGTATCTTCTGAGCAGATGGGTAAAGAAATTGGATAAGAATTCAACGTGGTGGCTGTTGAAAGTCATCACTGGGTCTTCTTGGTCCAGCCATTCCCTTTGGTCTCTGTCTTTGGCCTGGACCACCATCTTACCTCCATCATTCTTGAAGGACACACCTGAAGCCTCACCATTGCAGCAGGCTTATCATCAGGAATTAAT AAGGTGCAGGGAASEQ ID NO: 54 AAAAAACTGCTATAATTCAACCAGTTCTGACACAAATGGTGGGTTTCAGAGAACGGAGTAGAGATGAGAGAAGTGGCTTTTAGCAAGAAATTACGCAGGTGACTTTCAAATAAATCATCTACATAGAAGGCACGTTTTGCCTCTAATGTGCAAATAATTTAGCATCTGAAATGGCATGTAAAGATCTCATTTTAGATTCAATTCCACTCTAATTCATCAAAATGGGAAGTAAGGCATTGTGGCATAACAATTGGGATCAGAAGACCTGTGGTTACATCTCAGTTCAGCCCACAGAACTAACCGGGACTGAGGCCATTGGTCTTAGACCTCTCATCTGTGGGATGGGGTTACTAAGACTGATTTCCTATGTTTCTTTGCAAAACTTGAAATAATTGTGTGAAGAGTACTTTGCAAAGAGATAAGTGCTACAGAACTATAAAGTACTAAATAAAAGCTAATATATTAGGCATCTAATAGGTATTAATAGCAGCAAGGTCTGACCACAAGCAAACACCTATTTTAGGCACTGACTTCTGCCAAAAATCCGGAGCTAGATGGAGGAAGGAAAGGCTCATAAATCAGTCTCCT TAAGAGGCCTCCTSEQ ID NO: 55 CAGCTGGGGCAGGAAAACAACGGTGCTGCCTATTTCTTGGACCCCAACTGTTTCTAATTTGAGTCACTCTTCAGCACTCCGTTAAACCCCAGTCCCCATCCTCAGTTCCCAGGTGGGTTTGCATAAGGGGTAGTAGACCTGCCAGGAGATGCAGTGAGAAGCACTGAGCACTGAGGCAGGAGGAGCACAGACAGCACAGAGAATCCTCTAAAGTGAAGTTTCTCTCTTTCTCACTGTTGTTGGGGACTCACCCTGCACCACAAAGAAGACTAGATAAAGAAAACCACTAACCAGTAACATCCGGCTCAGTAGGCTGAGCACAAAAGCAAGGTTTTGCTTTGGCCCTAGACCTAGAAGCCTGCTGAGTGAGCGCCTTGGCCCTAGAAAAGAGGAGCAGGGGGTTGTCCGGAGCCCCTCCGACCACGCTGCTCTCAGTCAGGCACCACAGACACAGGCAGAGCTGGCTAGAGGTCAGAGGCATGGCCCCAGTGAGAAGCAGCAGCTGTGCCCTGTGAGCAGGTAGGATGGGGTTAAGGGCTGGCCTGGAATGGGGGCAGAAGGTGGAAGTTGGGAAATGGTGATGACAGC AGTTATTGCAAGGSEQ ID NO: 56 TGGCTTATTTTTAACTCACTTAATATTTTTCTCTTGAGAAATGTTAAGCTAATTTTTTTTTTTTTAAAGGGAAGCAAAAAGGCCTGTACTACTGTCAAGGCAGACTGAACTTATTTTATTGACTGTGTTTTTCGTTATGGAGGACTGTGTGTGACAGTACTTCTTTTACTTACAAAGAATTCTTTGTCACAAAAGCCTCATTCATCCACTGACATTTTGGGGTTCCTTTTTCTGTAAACAATCAATCTAAAAAGGAAGTGCCTAATCGGGTGTTTATGTTACAGGTGTAGCCTGGCTGGGCCGGGGGTAGTTGGCTTCCTTCTGTTTTTCCTATGCTCTCGGGAACATTTGACCTTTAGTTCCTTTCTAAATGCCTCAGTTCATTAGAAATGTTTTGTGCTCATTCAATAAGTCATTTATTTGAGGTCAGATTTATATTTTGAAAAGGTCATTTTGGCAAGTTATTTGGTCTTCCCAAAGTCCAGAGTGTCCAGTATAAAATAGGGATATCATTCCTATCTGTTTCCTATAATTGTTGTGAGGATGAATGTGTTAATGTAAGTGAAAATGCTTGGTAAACAAGAAGGA GATATATGTATTASEQ ID NO: 57 GATGTAAAAATGGGATCCTTGATTTTTAGTATCTGTTTTGCCGTCCAGCTGTGCTTGCTTTTTAGGCCCTAAAAACTGTATGTTTTCTTGGCCCTGTTACTTAAAAGGCTCTACCCTGAAGCCAGTAATCCAATTAACTTACTTCTTTAAGGAAATGTTTATATGTAAGGGTGTCTGCTTTTCCTCTCCATCTCACCTGAACTTTCACTCACGCCATTTTTCCTTTGTTTGCATAAAATATAAATTCTATATCTTGTTTCACTTAAGTTTGTCTCTTCAGAAATGCAGATTTAGAGTTGCCCGGCTAGGAACTGTTTATGGCAGGAAGCAGGTATTCAAGAGACTGTTGGTCTAAAATGGGGAAGAGAAGCTTAAAACTAGCAATTGAAAAATCTTTTGTCTGTCTGTGTAGGTTGTGTGTGTGATGTTTCCCTACTGAAATATATAAAGGTGCTCTAATTAATTGGCTTTAAAAAAATAAGCACTTACATAAAATATTTTGTCAGAAAAATAGAAACTAAAATGCCTTTTAGCTCAAGTGACTTTAATAATCTTTAGTAAATAAAAATAGTTTTACAATTATTGACA AAAGTCTTTAAAASEQ ID NO: 58 GACATAGTGATAGGTTAGACTGTCTATTGTGAGATTCACAAAGGGGTTTGGAACAGAAATCTAGACCAAGACCTTTTACACAAGAGATTCTTTGTGGAATACTTGCTGGAATATTCCCAGAGTATTTTGGGATACCCCAGACTCATACAAGGTTGTATAACCTCTAACTAACAAGATTCTAAGCAAAGGATGAGACGTGATCATGCTCAGGTAACGAAAACTTAGGGTCAATTCATGTTCTAATTAGTTCTCTTGATCTAACAGTTACGGCTTATAAATCATTCCATGTCGGAAGCCCCACCGGAAATAGTTGAGGTTCAATTCAGTTACAGCTCAATGAAGTCTGAAAACAAGTGTCCAACATTTTCTGGTTCATGGTTTAAAATAGGTTTCAAATAAACAATGAGGAAGCCAGTTTCCTGTTTGGGTTGGGTCCATTGGATCCTAGCCCATCAAAGCTTTGAATTATATTACAATGACAGGCAAGGACTAGAGGGGGAAGAACTGAAACGCAGAGAAAAGTTGGCACAGTGCCAGGAAACCTGGCTAAAATTAAGTCCCTCAGTCCAAAGAAAACAATGGCAGCTA GGACATGAGTCAAIn some embodiments, the methods disclosed herein utilize at least 15,at least 20, at least 30, at least 40, at least 50 or at least 60nucleotides of these nucleic acid sequences.

The following nucleic acid sequences are more methylated in fetal DNAthan maternal DNA, and can be used to detect fetal aneuploidy ofchromosome 21. In some embodiments, the methods disclosed herein utilizeat least 15, at least 20, at least 30, at least 40, at least 50 or atleast 60 nucleotides or all nucleotides of at least one of the followingnucleic acid sequences:

SEO ID NO: 59 AGACACACTTAAGCTGAGCTTAGAGCAAAGCATGTCCCTCAAGGTGACCTTGCCGCATGGCCTGAACCTAGAATTCCCCTGACCAGGGTGGACAGCCATCACCATGGGCCTTTCTGCCTGGCCTAGTATGGCTCTCCTTCGAACTCACCTAGACCCTTGCATCAAAGGACTCGGGGACTCTGCTGCAGTCATCCTAGCACGGTCGAGAGGAGTAGGAAGGTGCCCAGCGGCTCAGCCTTTAAGGAGGTCTATTGGTCACCATTGCTTCTGCCTTCTTGAGAACCATCACCCTCGGAGGATCCGGTAGTGTATCCCAGTCAAGTGTCAAATGCGTTGTTCCCTGGAGAGGAAGGCTTATTTCCAGAAAGTACTCTGACGTTTTGTAATTTGTGATTTCTATCACTGGCTCAGTGGCCTACCCACTGCTCAGCTCATGTGGCATGACCAGGCTGGGGCGTCGGGTCATCTTGTTCTCTGGGTGTGTGGCAACACTGGTTTATGGCGTAATTTCCTTCCTGAGAAGAAATTCAGTAATTTCTAATATTCAGGGCTTTAGGTAGAGGCCTGAAAATCACTCTCCATGCCTCT TTCAGTATTTTATSEQ ID NO: 60 TAGTAAGCAAACAACTGAGGGACCAAAAAAAAAAAACAGGTTTTAAAGTGACAAGGTTCAAATCTAGGGTAACAAATGGCAAGATGGGAGTACTCACATGATCAAAGAAATGTACCACTGCAAGCTTTTTGCTGCGCCTGGTAAAGATGCGCTGCACTTTAGCAATTTTGCCAAAATGGTTCTCCAGAATGGTCCTGTCGTTGAGGTAGTCAGGGATGTTCTTGCACTGGATGGCTGTGACTTCAGAGGGAGACAAGCCCCCAAGACTGTCTGTGCTCTCGCTTCTGTTACTCTGACGCGCCGGAGTTCCTCTTAGAGAATCTAGGGGTTCAGAGAATGGACACTTAAACCATCAGATGTTTCCAACAACAGGAGGCACCAACTGTGTCGAAGCAGAGGAAATTTTCAGATGTAAGACCTGCTCCGATGACATGTTAGAATCCCTAATGTTTATGGGAGATTTATCTGAAAAATCAAGCTAACACCCAGAGAAACGTATTGAAAACCTGCCTGCAGGATTTACTCTATGTGCTACATTTTCAGAGCTCACCTTCTTTCTTCTCTCTACTTTCAGTTTCTTCCTCTTTA TTCACACCTGGAASEQ ID NO: 61 AGAAGGCGCTTATACATCACGGCCGTTGCTCATGCTTGGGTTAAAAAAGGTCTCCCTTACAGCCAGGGCTCAGGATCCCTAGGCAGCTGGGGGTTGGCCCCCGGGGCTCAGAGGTTAACCCTCTTCCTCTCACAGATGGGCCTAGCGGTGGGAGCGTTTGTGGCAAGCTGCAGAAATGAGATGCGAGAGAGAGAGCCCGTCCTTCAGAGTCCACGACAGCACAAAGGAAATGGCCGTGGGTGTTTCGGGTTTAGCTGTCTTTGTTCCTACACGTTTATGTTACCTGCAAGTGGAAATTCACCGGCACATCCTCTCAAAAATTATATTTTGTGTAAAAATATTTAAGCACTAAATGAACCTCTGAGAGTCGGGATAGCGCTTCCTTGGAGGGGGGAAAGTTTGGGGAGGAGGGCACGGGGGCTCCCTTCCTTGGAGGGGAGAAAGTTTGGGGAGGAGGGCATGGGGGCTCCCTGAGGGCCAGTCATGCTGTTCTTGGTGTGGGTGGCAGTGACACAGGCGTTTGCTTAGGGACAACTGTGCTGTTCTGACTTAAGTGCCAGCCTGTGGAAGCGCCCACTGACTCAAGAT TAGATAATCTCCCSEQ ID NO: 62 GGATACAACTAGCCTAGAAAAAAGTCTTCAAAGTTCTTCTAAGCATTAGTTCTCCAAGTGCTCCAATGAAGGCTAACAACCCAATCCAAAGTGAAGGCTTCTTTTACTTCTTATAAACAATTTCATAAATTTTTGTCTTAGTATATAATAAAAGGATAATAAGCTACAAGATAAATGATTAAAGATTAACCAAATTTGGTATTCATGAACTAAAACGAACACTTTTCTAGATTTTAAGGTGGGCTCTACTTCAGCTCATTACTAAGATGACTAAGCACCATGCAAAATTTGGCAATGCTGCCGGGTCTCAAGCATATATATGAAAAAGTCTGCAGCCACAACTACCCTAACAGAGATTTCAGAAAGGATGCTCTAAGTCTATCATGAGTGTAACCTTAAATAGATTACTTTTATACTGTTTTATTCTTCCTTTTAAACTGTAAACTATATTAGTTTAATATCATCAATTTATAGTTAAGTTAGTTATTTGGTTAACTCTACAGTGTAGAGCTATGCATATTTGTTGTCTGCACCTTTAACTTTCCAGTAACTAAATGTTACGCGTTCGGTGTTGGAGAAATGAGACAT CTACTCAGTAACTSEQ ID NO: 63 AAACGGACCACAACTTTCTTCAAACTTCTTCAAAGTTTGCATGTCTGGGAGACCAATAAAGGGCAGGGCATACGAAGTAAGTCTGTTTAGGTCAGTTGTTTATGGGTAGGTGCGACTTCATTCCTGTTCGCCCTACTTGCAGACATGGTAACTTAGAACAGTTCTTCAGAAGCTGTTCGATGGCAACGTGGCGGACATGGAGCTGTGAGGCCAAAGAATCTTTTTCTTGCACTTGGTGAGTTAACTCTTCAAAAACTTCTGTAAAAGATTTAAAAACTTTCATTTTTAGTATGCAGAAAGCCGGTGCTATGATTTATATTTCAAGAACTCTAACTGAAATCTATGCATTGGGCAAAGTGTCACAATGAAACTGATTTGATACCAAGGAAACAGTTCTGTTCTCTACAAAGCATATCAGTTCTGATCATCCTAGGCATCTGTATACTTTACATAGCCAAATGGAATTTGGCTCTTCTCCATCAAGTGACTACAGAGATACACAGCAAAAATGGTGGTTGCTACTGTAGCTGAGCTACTTTTCTCAATTAGAATCTATAAACAGATTCAATTAGTACATAATGTGATTTA TGGACAAGAAGTGSEQ ID NO: 64 CTGCATTTTGTGTGAGTATAGCTGGATCCGCTTTGATTCTACAAATTAGGTGCCCTTCTGTTTCTTAGTAATGTCATATTCTCTGACCACTGTAGCCACTTAAGATAGTCTCAAATATGAATAATTTTCAGTGGCCATAGTGGAGAAGATGCTGACCCAGGAGGCTTATTCTCACCTCCACCTCTTAGACTCACATGTATGATTTTTTTTTCTCCCTACCAAAGTGGCTAGTTTTGACAGCCTTTAGGTTGCAGAACTGTTTATATTCGAAAGAAGACCTTACCTTCATAGAGCATTTTCCCGGGCAGTTGTTCTGCTTTGTGTTTTTAATATCTACTCTTATTCGGAGTCCCAAAATGAGTTAGCTTGGGTAGCTCTCACAATGAAAACTGATTTCTTCGCATAAAACTTTATTCTACCTTTTTGAAAACCAGCTGGAAATGATGAATGGTGGCTTCAACAGTAATGGTTCACGAAGAACTGATCGTGTCCCCATCAATGAGGTCTTACCCATGGCAGGGCAATGGCGGTCTACTGTAATTCTGTGTTGGTTGTAGTAAGTTGGGGGAATATCAGGCCAAGAATCAA CAGAGCTACGTGGSEQ ID NO: 65 TAACCCTATCTAGTACAAGTTCAATTATGTTAAGCTAAAAACTTTGCTGTAAAATATTAAATGCCAAGCTCAAAAAATCATAAGGGACATCCTCTCAGCCCTAAGTTCTATTCACCCTGATACTCAATACATTTGAAAGTAAAACCTCACTTTCGAAGTAACGCTGATACTGAAGCAAGGAGATGTTGAATGACTCACCCATTTCATAGCTTTTCCGTGGCAAAGCCTCACTAAAACCTATCTAATCCCTAGCTCCAATTCTTTAATCTCCATGGTGCTTCTGATTAATAGTTAAACCACCCGGAAGTATGTTAAGGATACTAACATTTTCACCCCGAAGTATTATCCTTCGCCATCCTCTAGAATATGCTATGCAAAGAGGCCTAAAACAAAATAAACTGCGACTTCAGCAGCTGAGAAGTAATGATTCCTTTCCCCTCCCACACATCAAAACTTCCTATCCTATCCCCTCACTACTTTTTTTCCTTTTTAAAAGTAACAATGGTCATTGAGAACTACTAAATCCTGCAACACTCACAAGGTAGTATGTACAACCACAGTTTTCCCCCGCGATACTTCACTGAAGTA TTCAATACAGCCASEQ ID NO: 3 TGATAACACTGGGGAGAGTTAACTTTTTCACCTCTGTTTGTTGATATGCCCCTTTGTGCTCCGTATTCACAAAGGGGCACCTCAACAAGCCCATCATTTGTTAATGAATGCCTTTACTTTCATCGCCAGCAAGACATTTTCATAAACCCACCATCTACTTTGCAGTTCTCAAGAGTTGCTTCTCTTAAACTGGTGAGGACGCGGCTAAGCCCTGGAAATGAGTCCAGATTCTCACGGTGGCCCATAAACACTGCTGTTTCCTCCCACCTAGGAAACCCGCAGACCTGCAGAACCTGGCTCCCGGAACAAACCCTCCTTTCATGACTTTTGATGGTGAAGTCAAGACGGATGTGAATAAGATCGAGGAGTTCTTAGAGGAGAAATTAGCTCCCCCGAGGTAGGCCTCAGAAAACCAGTGTTCATAACTTGATTGTCACTTTCCCCCACTAGTAGTCAGTTCTAAAGCTCTGGGCAGTGTGTGTGTGTGTGGTTTTCTGCAGCCCACGGTGCTCACTTCCTTAATCATAACCCTGGTGTAACCAGATTAGAGTTCGGGGACCTGGGTTTCATTGTGCTGCACCTGCAGCT TGGCAATCACAGTSEQ ID NO: 67 GCAAACCACCCACTTCTGCCCTCTGCCCTTCTTCCCTTTTCTCGACACCCTGCGGCCCCCCAGTTTCAGCAGAGTTTATTTGGGGTGAAAAACAAGAGATGCTCAGCGCCTGTGGGATGTGTGGGCTGACTCGTACATTAGGATGTGTGTCAATCTGAAATAACCTGGCCGTTATATGGATGCCTTGGGGCTTGGGGGGTTTCTGGCAGTCTGTCGAGCCCGAGGTGAATGTCCCCAAGGCTGCTGGTGAATCAGATCCCTGGCGTTCTCCGTTGGCAGTTCAGCCCAACAGTTTCTCTGCCGGCCGTGCCTCTGCAGGTCCCTCCTCTGATCTGATTGGATTAATATTTGAATCAATAGACTGAGTCAAGCAGAATGTGGGTGGGCCTCATGCAATCAGCTGAAGCCCTGAAAAGAGCAAAAGGGCTGCCCCTTCCCCCGAGGAGGAGAGAACCCCTCCTGCCGGACGGCCTCCGAACTGGAACATCAGCTTTTTGCTGCCTTTGGACTTGAACTGAAACATTACCTCTCCCTGGGTCTTGAGTCTGCCTACGTTGGCACGGGAACTACACGTTGGCTCTCTTGGGC CCCAGCTTGCTGASEQ ID NO: 68 CCTTTGTTCTTGTTGGCATCTGTGTAAGAGAATCCAGGGCCTGAACCATTGTCCACTCAAACAGACCATACACACCTGGTCCAGTTTTGTGCATGCCTCCCTTTTCCACAGTGTGCCACTTGCTATAGTTCTGAACAAAAACTCCCTTGCCCTTCGGAGCTTTATTTATTTTTAATTTGCTCTGTCATTGCATTGCATGCAATAGAAGCTTCCTGGTGGAAGCTCAATGTTCTGCTCAGTCCCAGACACGTTTTCAGCCACTGTATCATTGCCTTAGGTTGTGGTTTCCCCAAAGCAGCACCGGAGGTAAGGGCTTGTGTGCAGGAGTTCACTTGGGAGGTGGCTCTAGGAAATAGAAGCGAGTTTCCAGGAAGTGTGGGATGAATACGGGGATGGGGTGGGGGGCGGGGAGGAATCCAATATGAATATATAATCTCATATTGAGTATAAAAATCCAATATGAGTATGTTTTCCAGACTGCTGCTAAGGAGAATGGTGAATTATTCTACTGCGACCTTTTGAGGGTCCACACAATGCCTCCCAAAACTATCTACCAGAGGGTGGATAGGAAGCATTGATCCATGGCTT ATATTCCCCATTG

In some embodiments, the methods disclosed herein utilize at least 15,at least 20, at least 30, at least 40, at least 50 or at least 60nucleotides of these nucleic acid sequences.

All nucleic acid sequences from U.S. Provisional Application No.61/361,824 are incorporated by reference herein in their entirety.

In some embodiments, the methods disclosed herein use a portion, or theentirety, of one, more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 orall of SEQ ID NOs: 1-7, 29-38 and 59-68, and 83 to detect aneuploidy ofchromosome 21. In additional embodiments, the methods disclosed hereinuse a portion, or the entirety, of one, more than one, such as 2, 3, 4,5, 6, 7, 8, 9, 10 or all of SEQ ID NOs: 8, 19-28 and 49-58 to detectaneuploidy of chromosome 18. In some embodiments, the methods disclosedherein use a portion, or the entirety, of one, more than one, such as 2,3, 4, 5, 6, 7, 8, 9, 10 or all of SEQ ID NOs: 9-18 and 39-48 to detectaneuploidy of chromosome 13.

In some embodiments, multiple selected loci are analyzed in parallel.Without being bound by theory, this can be to reduce the negative effectof inter-individual variation in absolute DNA methylation level at anygiven locus. The resulting test can provide highly accuratedetermination of the copy number of a particular chromosome or region ofthat chromosome relative to the other chromosomes tested. Biomarkersspecific to chromosomes 13, 18 and 21, enable the test to define anormal range of inter-chromosomal count ratios between chromosomes 13,18 and 21 for euploid fetuses and thereby determine deviation from thisnormal variation in when a fetus is tested that has aneuploidy on eitherchromosome 13, 18 or 21. A copy number ratio of 1:2 or 2:1 indicates thefetus has aneuploidy.

In some embodiments, fetal DNA is isolated from a maternal sample, suchas, but not limited to, blood, serum or plasma. In addition to theacellular portion of the whole blood, DNA can also be recovered from thecellular fraction, enriched in the buffy coat portion, which can beobtained following centrifugation of a whole blood sample from the womanand removal of the plasma. There are numerous known methods forextracting DNA from a biological sample including blood. The generalmethods of DNA preparation (e.g., described by Sambrook and Russell,Molecular Cloning: A Laboratory Manual 3d ed., 2001) can be followed;various commercially available reagents or kits, such as the QIAamp DNAMini Kit or QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany),GENOMICPREP™. Blood DNA Isolation Kit (Promega, Madison, Wis.), andGFX™. Genomic Blood DNA Purification Kit (Amersham, Piscataway, N.J.),may also be used to obtain DNA from a blood sample from a pregnantwoman. Combinations of more than one of these methods may also be used.The methods disclosed herein use the methylation status of fetal DNA toisolate fetal DNA from maternal DNA. Once the fetal DNA is isolated, thecopy number of an allele on a chromosome of interest can be determined.

In some examples, a sample, such as a blood, serum or plasma sample, isobtained from a pregnant woman at a suitable gestational age, such as 10to 14 weeks, or 11 to 13 weeks for a human subject.

The gestational age may vary depending on the disorder to be assessedand the mammalian species. In some embodiments, a sample from the firstor second trimester of pregnancy is utilized. Collection of blood from apregnant female is performed in accordance with the standard protocolhospitals or clinics generally follow. For a pregnant woman, anappropriate amount of peripheral blood, such as between 5-50 ml, can becollected and may be stored according to standard procedures.

The analysis of fetal DNA found in maternal blood can performed using,for example, whole blood, serum, or plasma. The methods for preparingserum or plasma from maternal blood are well known among those of skillin the art. For example, a pregnant woman's blood can be placed in atube containing EDTA or a specialized commercial product such asVACUTAINER® SST (Becton Dickinson, Franklin Lakes, N.J.) to preventblood clotting, and plasma can then be obtained from whole blood throughcentrifugation.

Serum can be obtained with or without centrifugation following bloodclotting. If centrifugation is used then it is typically, though notexclusively, conducted at an appropriate speed, such as 1,500-3,000 timegravity. Plasma or serum may be subjected to additional centrifugationsteps or purification steps before being transferred to a fresh tube forDNA extraction.

There are numerous known methods for extracting DNA from a biologicalsample. General methods of DNA preparation (see, for example, Sambrookand Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001) can befollowed; various commercially available reagents or kits, such asQIAAMP™ DNA Mini Kit or QIAAMP™ DNA Blood Mini Kit (Qiagen, Hilden,Germany), GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wis.),and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway,N.J.), can also be used to obtain DNA from a sample, such as a blood,serum or plasma sample, from a pregnant woman. Combinations of more thanone of these methods may also be used.

The DNA present in a sample from a pregnant woman, whether or notextracted from the sample, is then treated with an agent capable ofpreferentially modifying DNA depending on whether the DNA sequence ismethylated. For instance, this agent can be an enzyme that digests DNAin a methylation sensitive manner, for example only unmethylated DNAwill be digested while methylated DNA remains unchanged. Another methodincludes the utilization of an agent that selectively converts apolynucleotide sequence depending on the methylation status. Typically,such an agent reacts with the unmethylated C residue(s) in a DNAmolecule and converts each unmethylated C residue to a uracil (U)residue, whereas the methylated C residues remain unchanged. This C to Uconversion allows detection and comparison of methylation status basedon changes in the primary sequence of the nucleic acid. An exemplaryreagent suitable for this purpose is bisulfite, such as sodiumbisulfite. Methods for using bisulfite for chemical modification of DNAare well known in the art (see, e.g., Herman et al., Proc. Natl. Acad.Sci. USA 93:9821-9826, 1996). These methods can be used to selectivelypurify fetal DNA from maternal DNA.

Additional methods for identifying and/or purifying regions ofdifferential methylation and for determining the allelic ratio, aredescribed in, for example, U.S. Pat. No. 5,871,917; U.S. Pat. No.5,436,142; and U.S. Patent Application No. US20020155451A1, U.S. PatentApplication No. US20030022215A1, U.S. Patent Application No.US20030099997, and U.S. Patent Application No. 2009/0019278, thecontents of which are herein incorporated by reference in theirentirety.

In one embodiment, the method utilizes a restriction enzyme. Examples ofsuitable restriction enzymes for use include, but are not limited toBsiSI, Hin2I, MseI, Sau3A, RsaI, TspEI, MaeI, NiaIII, DpnI and the like.One methyl-sensitive enzyme is Hpa II that recognizes and cleaves atnonmethylated CCGG sequences but not at CCGG sequences where the outercytosine is methylated. In this manner, a restriction enzyme can beselected that cleaves maternal, but not fetal DNA, or that cleavesfetal, but not maternal DNA. The difference in methylation betweenmaternal and fetal DNA can be assessed and used to isolate fetal DNA bybisulfide treatment followed by either 1) sequencing, or 2)base-specific cleavage followed by mass spectrometric analysis asdescribed in von Wintzingerode et al., 2002, PNAS, 99:7039-44, hereinincorporated by reference in its entirety. These methods generally allowthe detection and/or amplification of fetal DNA. Thus, in someembodiments, the methylation status of the maternal DNA is used toselectively degrade maternal DNA. For example, maternal DNA can berestricted by an appropriate enzyme and degraded, or bisulfite treatmentcan be utilized. Any method that selects for fetal DNA can be utilized.

Following the methylation-dependent isolation of fetal DNA, one or moreof the relevant nucleic acid sequences (such as at least 15, 20, 25, 30,40, 50, 60 or all of the nucleotides of at least one of the nucleic acidsequences set forth as SEQ ID NO: 1-68 or 83) from the fetal source maybe distinguished from their counterparts from the maternal source. Insome embodiments, the allelic ratio is determined.

In some examples, a single nucleotide polymorphism (SNP) that is locatedadjacent to the CpG containing sequence, is utilized. For example, theSNP can be at most 1,000, at most 150, at most 100 or at most 50 basepairs from the CpG containing genomic sequence in the maternal (and/orfetal) genome. In other embodiments, a STR that is located adjacent tothe CpG containing sequence is utilized. For example, the SNP can be atmost 1,000, at most 150, at most 100 or at most 50 base pairs from theCpG containing genomic sequence in the maternal (and/or fetal) genome.

In additional embodiments, a nucleic acid including a SNP, such as abi-allelic (heterozygous SNP) can be used to detect the copy number ofchromosome 13, 18 or 21 in the fetus. In some embodiments, the SNP is abi-allelic SNP that is within 150 base pairs, within 140 base pairs,within 130 base pairs, with 120 base pairs, within 110 base pairs,within 100 base pairs, within 75 base pairs, within 50 base pairs orwithin 25 base pairs of at least one of SEQ ID NOs: 1-68 or 83, and canbe used to detect the copy number of chromosome 13, 18 or 21 in thefetus. Generally, the number of base pairs is measured from the 5′ endof SEQ ID NO: 1-68 or SEQ ID NO: 83, or the 3′ end of SEQ ID NO: 1-68 orSEQ ID NO: 83, depending on whether the bi-allelic SNP is located 5′ or3′ of SEQ ID NO: 1-68 or SEQ ID NO: 83, respectively. In additionalembodiments, the bi-allelic SNP is identified using a heterozygositycut-off of about 0.25, such as 0.25.

In further embodiments, the copy number of a short tandem repeat isdetermined in order to detect fetal anueploidy. Short tandem repeats(STR) are also bi-allelic in karyotypically normal individuals and mayor may not be tri-allelic or mono-allelic in individuals withaneuploidy. Thus, the disclosed methods can also determine the copynumber of short tandem repeats. In additional embodiments, a nucleicacid including a STR, such as a bi-allelic, tri-allelic or mon-allelic(heterozygous STR) can be used to detect the copy number of chromosome13, 18 or 21 in the fetus. In some embodiments, the STR is within 150base pairs, within 140 base pairs, within 130 base pairs, with 120 basepairs, within 110 base pairs, within 100 base pairs, within 75 basepairs, within 50 base pairs or within 25 base pairs of at least one ofSEQ ID NOs: 1-68 or 83, and can be used to detect the copy number ofchromosome 13, 18 or 21 in the fetus. Generally, the number of basepairs is measured from the 5′ end of SEQ ID NO: 1-68 or SEQ ID NO: 83,or the 3′ end of SEQ ID NO: 1-68 or SEQ ID NO: 83, depending on whetherthe STR is located 5′ or 3′ of SEQ ID NO: 1-68 or SEQ ID NO: 83,respectively. In additional embodiments, the bi-allelic, tri-allelic ormono-allelic STR is identified using a heterozygosity cut-off of about0.25, such as 0.25.

An amplification reaction is optional prior to another analysis, such asa copy number and/or sequenced based analysis for a fetal marker(including, but not limited to, a single nucleotide polymorphism, asdescribed above, or one or more of SEQ ID NOs: 1-68 and 83), aftertreatment by the methylation-dependent differential modificationprocess. In some embodiments, the amplification is performed topreferentially amplify a fetal marker such as such as at least 15, 20,25, 30, 40, 50, 60 or all of the nucleotides of at least one of thenucleic acid sequences set forth as SEQ ID NO: 1-68 and 83, or a nucleicacid including a SNP, such as a bi-allelic SNP that is within 150 basepairs, within 140 base pairs, within 130 base pairs, with 120 basepairs, within 110 base pairs, within 100 base pairs, within 75 basepairs, within 50 base pairs or within 25 base pairs of at least one ofSEQ ID NOs: 1-68 and 83, or a short tandem repeat.

A variety of polynucleotide amplification methods are well establishedand frequently used in research. For instance, the general methods ofpolymerase chain reaction (PCR) for polynucleotide sequenceamplification are well known in the art. Reviews of PCR methods,protocols, and principles in designing primers, are provided forexample, in Innis, et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, Inc. N.Y., 1990, which is incorporatedherein by reference. PCR reagents and protocols are also available fromcommercial vendors, such as Roche Molecular Systems.

PCR can be carried out as an automated process with a thermostableenzyme. In this process, the temperature of the reaction mixture iscycled through a denaturing region, a primer annealing region, and anextension reaction region automatically. Machines specifically adaptedfor this purpose are commercially available. Although PCR amplificationof a target polynucleotide sequence (e.g., that of such as at least 15,20, 25, 30, 40, 50, 60 or all of the nucleotides of at least one of thenucleic acid sequences set forth as SEQ ID NO: 1-68 or 83, or a SNP orshort tandem repeat within a certain distance of at least one of thesesequences) is typically used in practicing the present invention, one ofskill in the art will recognize that the amplification of a genomicsequence can be accomplished by any known method, such as ligase chainreaction (LCR), transcription-mediated amplification, and self-sustainedsequence replication or nucleic acid sequence-based amplification(NASBA), each of which provides sufficient amplification. Branched-DNAtechnology can also be used to qualitatively demonstrate the presence ofa particular genomic sequence (see Nolte, Adv. Clin. Chem. 33:201-235,1998).

In some embodiments, the fetal DNA is sequenced. Techniques forpolynucleotide sequence determination are also well established andwidely practiced in the relevant research field. DNA sequencing methodsare routinely practiced in research laboratories, either manual orautomated, and can be used in the methods disclosed herein (see forexample, Sambrook and Russell, Molecular Cloning, A Laboratory Manual(3rd ed. 2001); Kriegler, Gene Transfer and Expression: A LaboratoryManual (1990); and Current Protocols in Molecular Biology (Ausubel etal., eds., 1994)). However, there are additional protocols suitable fordetecting changes in a polynucleotide sequence, or for determining copynumber, that are of use. These methods include, but are not limited to,mass spectrometry, primer extension, polynucleotide hybridization,real-time PCR, and electrophoresis. The methods can include short readDNA sequencing, pyrosequencing, real time PCR or single moleculesequencing (for example, Pacific Biosciences methodology).

The presence and quantity of the fetal nucleic acids can be determinedand compared to a standard control, such as the presence and quantity ina maternal sample, the presence and quantity in a fetal sample from afetus known not to have aneuploidy, the presence and quantity in a fetalsample from a fetus known to have aneuploidy, or a standard value.Furthermore, once it is determined that one or more of these nucleicacids is of fetal origin that is indeed present in the sample,particularly when the amount of the gene(s) is greater than (or lessthan) a pre-determined threshold, the sample and its equivalents aredeemed to contain a sufficient amount of fetal DNA for further analyses.The quantity of these particular nucleic acid sequences can be measuredas fetal markers indicative of certain conditions.

In several embodiments, an aneuploidy of a chromosome, such aschromosomes 13, 18 or 21, is determined by assessing the copy number ofthe target polynucleotide sequence. This can be done using any methodthat quantifies changes in the allelic ratio. In several examples,MASSARRAY® by Sequenome, pyrosequencing and TAQMAN® (digital format),sequencing, hybridization, padlock (inversion) probes can be utilized. Acopy number that is significantly different from 1:1, such as a copynumber of 1:2 or 2:1 indicates that the fetus is anuepolid. In someexamples, the target polynucleotide sequence is a 15, 20, 20, 40, 50 or60 nucleotide sequences located within any one of SEQ ID NOs: 1-68 or83. In other examples, the target polynucleotide sequence is SEQ ID NO:1-68 or 83. In further examples, the target polynucleotide sequenceincludes a target SNP, such as a bi-allelic SNP that is within 150 basepairs, within 140 base pairs, within 130 base pairs, with 120 basepairs, within 110 base pairs, within 100 base pairs, within 75 basepairs, within 50 base pairs or within 25 base pairs of at least one ofSEQ ID NOs: 1-68 or 83. Thus, the SNP can be at most 150 base pairs,within 140 base pairs, within 130 base pairs, with 120 base pairs,within 110 base pairs, within 100 base pairs, within 75 base pairs,within 50 base pairs or within 25 base pairs from one of SEQ ID NOs:1-68 or 83. In more examples, the target polynucleotide sequence is ashort tandem repeat that is within 150 base pairs, within 140 basepairs, within 130 base pairs, with 120 base pairs, within 110 basepairs, within 100 base pairs, within 75 base pairs, within 50 base pairsor within 25 base pairs of at least one of SEQ ID NOs: 1-68 or 83.

Exemplary SNPs are provided below. The Rs. Id number and positioninformation is provided; sequence information for these SNPs isincorporated by reference herein as available on Jun. 30, 2010.Information can also be found at the NCBI Single Nucleotide Polymorphismwebsite. The SEQ ID NO: 2 that can be used with each SNP (see above) isindicated.

The following exemplary SNPs can be used to detect fetal aneuploidy ofchromosome 21:

Sequence 42 chromosome 21 (SEQ ID NO: 1)

Rs.id 914232, snp.pos 45777178 (snp150_c21_mc_flank.csv)

Sequence 45 chromosome 21(SEQ ID NO: 2)

Rs.id 12627387, snp.pos 42355995 (snp150_c21_mc_flank.csv)

Sequence 16 chromosome 21(SEQ ID NO: 3)

Rs.id 225395, no snp.pos (snp150_c21_cm_flank.csv)

Sequence 69 chromosome 21 (SEQ ID NO: 4)

Rs.id 2013275, snp.pos 45158900 (snp150_c21_mc_flank.csv)

Sequence 76 chromosome 21(SEQ ID NO: 5)

Rs.id 2255526, snp.pos 46795967 (snp_(—)150_c21_mc_flank.csv)

Sequence 88 chromosome 21 (SEQ ID NO: 6)

Rs.id 1539757, snp.pos 31559784 (snp150_c21_mc_flank.csv)

Sequence 99 chromosome 21 (SEQ ID NO: 7)

Rs.id 2838434, snp.pos 44161580 (snp150_c21_mc_flank.csv)

The following exemplary SNPs can be used to detect fetal aneuploidy ofchromosome 13:

Row 2 (SEQ ID NO: 9)

Rs.id 9542537, no snp.pos (snp150_c13_cm_flank.csv)

Row 4 (SEQ ID NO: 10)

Rs.id 7983181, no snp.pos (snp150 c13_cm_flank.csv)

Row 6 (SEQ ID NO: 11)

Rs.id 166710, no snp.pos (snp150_c13_cm_flank.csv)

Row 8 (SEQ ID NO: 12)

Rs.id 9301803 or 9301804, no snp.pos (snp150_c13_cm_flank.csv)

Row 12 (SEQ ID NO: 13)

Rs.id 2025675, no snp.pos (snp150_c13_cm_flank.csv)

Row 14 (SEQ ID NO: 14)

Rs.id 11617606, no snp.pos (snp150_c13_cm_flank.csv)

Row 16 (SEQ ID NO: 15)

Rs.id 9535813 or 9316563, no snp.pos (snp150_c13_cm_flank.csv)

Row 20(SEQ ID NO: 16)

Rs.id 980094 or 2389355, no snp.pos (snp150_c13_cm_flank.csv)

Row 24 (SEQ ID NO: 17)

Rs.id 9536376, no snp.pos (snp150_c13_cm_flank.csv)

Row 26 (SEQ ID NO: 18)

Rs.id 9572623, no snp.pos (snp150_c13_cm_flank.csv)

The following exemplary SNPs can be used to detect aneuploidy ofchromosome 18:

Row 2 (SEQ ID NO: 19)

Rs.id 12955286 or 1852531, no snp.pos (snp150_c18_cm_flank.csv)

Row 6 (SEQ ID NO: 20)

Rs.id 1244833, no snp.pos (snp150_c18_cm_flank.csv)

Row 8 (SEQ ID NO: 21)

Rs.id 2923220, no snp.pos (snp150_c18_cm_flank.csv)

Row 10 (SEQ ID NO: 22)

Rs.id 16977803, no snp.pos (snp150_c18_cm_flank.csv)

Row 12 9(SEQ ID NO: 23)

Rs.id 603884, no snp.pos (snp150_c18_cm_flank.csv)

Row 14 (SEQ ID NO: 24)

Rs.id 4800573, no snp.pos (snp150_c18_cm_flank.csv)

Row 16 (SEQ ID NO: 25)

Rs.id 12970409 (snp150_c18_cm_flank.csv)

Row 18 (SEQ ID NO: 26)

Rs.id 11663168 or 11663172 (snp150_c18_cm_flank.csv)

Row 22 (SEQ ID NO: 27)

Rs.id 7505859 (snp150_c18_cm_flank.csv)

Row 24 (SEQ ID NO: 28)

Rs.id 8095592 (snp150_c18_cm_flank.csv)

The following exemplary SNPs can be used to detect aneuploidy ofchromosome 21:

Row 2 (SEQ ID NO: 29)

Rs.id 225395 (snp150_c21_cm_flank.csv)

Row 4 (SEQ ID NO: 30)

Rs.id 2837528 (snp150_c21_cm_flank.csv)

Row 6 (SEQ ID NO: 31)

Rs.id 2827557 (snp150_c21_cm_flank.csv)

Row 8 (SEQ ID NO: 32)

Rs.id 2822564 (snp150_c21_cm_flank.csv)

Row 10 (SEQ ID NO: 33)

Rs.id 233895 (snp150_c21_cm_flank.csv)

Row 12(SEQ ID NO: 34)

Rs.id 9980448 (snp150_c21_cm_flank.csv)

Row 14 (SEQ ID NO: 35)

Rs.id 2827384 (snp150_c21_cm_flank.csv)

Row 16 (SEQ ID NO: 36)

Rs.id 20457173 (snp150_c21_cm_flank.csv)

Row 18 (SEQ ID NO: 37)

Rs.id 9977149 (snp150_c21_cm_flank.csv)

Row 20 (SEQ ID NO: 38)

Rs.id 3453 (snp150_c21_cm flank. Csv)

The following exemplary SNPs can be used to detect fetal aneuploidy ofchromosome 13:

Row 2 (SEQ ID NO: 39)

Rs.id 7317471, snp.pos 20518084 (snp150_c13_mc_flank.csv)

Row 4 (SEQ ID NO: 40)

Rs.id 3742160, snp.pos 105943830 (snp150_c13_mc_flank.csv)

Row 6 (SEQ ID NO: 41)

Rs.id 206321, snp.pos 31888780 (snp150_c13_mc_flank.csv)

Row 8 (SEQ ID NO: 42)

Rs.id 9579199 or 9578047, snp.pos 28062783 or 28062731(snp150_c13_mc_flank.csv)

Row 12 (SEQ ID NO: 43)

Rs.id 9506534, snp.pos 20145165 (snp150_c13 mc_flank.csv)

Row 14 (SEQ ID NO: 44)

Rs.id 1411551, snp.pos 109171036 (snp150_c13_mc_flank.csv)

Row 17 (SEQ ID NO: 45)

Rs.id 9515119, snp.pos 109207337 (snp150_c13_mc_flank.csv)

Row 19 (SEQ ID NO: 46)

Rs.id 9551454, snp.pos 27730987 (snp150_c13_mc_flank.csv)

Row 21 (SEQ ID NO: 47)

Rs.id 17593586, snp.pos 40693966 (snp150_c13_mc_flank.csv)

Row 23 (SEQ ID NO: 48)

Rs.id 166753, snp.pos 108544047 (snp150_c13_mc_flank.csv)

The following exemplary SNPs can be used to detect fetal aneuploidy ofchromosome 18:

Row 2 (SEQ ID NO: 49)

Rs.id 8083921, snp.pos 58917082 (snp150 c18_mc_flank.csv)

Row 4 (SEQ ID NO: 50)

Rs.id 546680, snp.pos 30988098 (snp150_c18_mc_flank.csv)

Row 6 (SEQ ID NO: 51)

Rs.id 4891159, snp.pos 72230929 (snp150_c18_mc_flank.csv)

Row 8 (SEQ ID NO: 52)

Rs.id 16978450, snp.pos 41517284 (snp150 c 8 flank.csv)

Row 10 (SEQ ID NO: 53)

Rs.id 7245283, snp.pos 957483 (snp150_c18_mc_flank.csv)

Row 12 (SEQ ID NO: 54)

Rs.id 9945379, snp.pos 10928309 (snp150_c18_mc_flank.csv)

Row 14 (SEQ ID NO: 55)

Rs.id 12958513 or 16978452 or 7228161, snp.pos 41520914 or 41521069 or41520883 (snp150_c18_mc_flank.csv)

Row 21 (SEQ ID NO: 56)

Rs.id 11152348, snp.pos 58349472 (snp150 c18 mc_flank.csv)

Row 23 (SEQ ID NO: 57)

Rs.id 528129, snp.pos 24748157 (snp150_c18_mc_flank.csv)

Row 25 (SEQ ID NO: 58)

Rs.id 16978485 or 9966818, snp.pos 41589920 or 41589897 (snp150c18_mc_flank.csv)

The following exemplary SNPs can be used to detect fetal aneuploidy ofchromosome 21:

Row 2 (SEQ ID NO: 59)

Rs.id 11702354, snp.pos 34806395 (snp150_c21_mc_flank.csv)

Row 4 (SEQ ID NO: 60)

Rs.id 11702450, snp.pos 46528077 (snp150_c21_mc_flank.csv)

Row 6 (SEQ ID NO: 61)

Rs.id 2839418, snp.pos 42192853 (snp150_c21_mc_flank.csv)

Row 8 (SEQ ID NO: 62)

Rs.id 6517531, snp.pos 39559517 (snp150_c21_mc_flank.csv)

Row 10 (SEQ ID NO: 63)

Rs.id 2824493, snp.pos 18087813 (snp150_c21_mc_flank.csv)

Row 12 (SEQ ID NO: 64)

Rs.id 2835676, snp.pos 37513181 (snp150_c21 mc_flank.csv)

Row 14 (SEQ ID NO: 65)

Rs.id 2823026, snp.pos 15346340 (snp150_c21_mc_flank.csv)

Row 16 (SEQ ID NO: 66)

Rs.id 6517254 or 2070368, snp.pos 35002160 or 35002268(snp150_c21_mc_flank.csv)

Row 20 (SEQ ID NO: 67)

Rs.id 220269, snp.pos 42357265 (snp150_c21_mc_flank.csv)

Row 22 (SEQ ID NO: 68)

Rs.id 2823304, snp.pos 15784966 (snp150_c21_mc_flank.csv)

In some embodiments, the methods disclosed herein utilize at least 1, atleast 5, at least 10, at least 15, at least 20, at least 30, at least40, at least 50 or at least 60 nucleotides of these SNPs.

In some embodiments, once a the fetus is diagnosed with aneuploidy, suchas a trisomy or a monosomy, an indication of that diagnosis can bedisplayed and/or conveyed to a clinician or other caregiver. Forexample, the results of the test are provided to a user (such as aclinician or other health care worker, laboratory personnel, or patient)in a perceivable output that provides information about the results ofthe test. In some examples, the output is a paper output (for example, awritten or printed output), a display on a screen, a graphical output(for example, a graph, chart, voltammetric trace, or other diagram), oran audible output.

Molecular Methods for Detection of Nucleic Acids

Polymorphisms, such as SNPs, also can be used to detect the allelefrequency both in the maternal sample and the fetal sample. Generally,the methods disclosed herein involve an assessment of nucleic acidsequence. Molecular techniques of use in all of these methods aredisclosed below.

Preparation of Nucleic Acids for Analysis:

Nucleic Acid Molecules can be prepared for analysis using any techniqueknown to those skilled in the art. Generally, such techniques result inthe production of a nucleic acid molecule sufficiently pure to determinethe presence or absence of one or more variations at one or morelocations in the nucleic acid molecule. Such techniques are describedfor example, in Sambrook, et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory, New York) (1989), and Ausubel, et al.,Current Protocols in Molecular Biology (John Wiley and Sons, New York)(1997), incorporated herein by reference.

When the nucleic acid of interest is present in a cell, it can benecessary to first prepare an extract of the cell and then performfurther steps, such as differential precipitation, columnchromatography, extraction with organic solvents and the like, in orderto obtain a sufficiently pure preparation of nucleic acid. Extracts canbe prepared using standard techniques in the art, for example, bychemical or mechanical lysis of the cell. Extracts then can be furthertreated, for example, by filtration and/or centrifugation and/or withchaotropic salts such as guanidinium isothiocyanate or urea or withorganic solvents such as phenol and/or HCCl₃ to denature anycontaminating and potentially interfering proteins. When chaotropicsalts are used, it can be desirable to remove the salts from the nucleicacid-containing sample. This can be accomplished using standardtechniques in the art such as precipitation, filtration, size exclusionchromatography and the like.

Amplification of Nucleic Acid Molecules:

Optionally, the nucleic acid samples obtained from the subject areamplified prior to detection. Target nucleic acids are amplified toobtain amplification products, including a DNA that includes a SNP, fromthe sample prior to detection.

Any nucleic acid amplification method can be used. An example of invitro amplification is the polymerase chain reaction (PCR), in which abiological sample obtained from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for hybridizationof the primers to a nucleic acid molecule in the sample. The primers areextended under suitable conditions, dissociated from the template, andthen re-annealed, extended, and dissociated to amplify the number ofcopies of the nucleic acid molecule. Other examples of in vitroamplification techniques include quantitative real-time PCR, stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see PCT Publication NO.WO 90/01069); ligase chain reaction amplification (see EP-A-320 308);gap filling ligase chain reaction amplification (see U.S. Pat. No.5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.6,027,889); and NASBA™ RNA transcription-free amplification (see U.S.Pat. No. 6,025,134).

In specific examples, the target sequences to be amplified from thesubject include a nucleotide sequence of interest including the SNP. Incertain embodiments, target sequences containing one or more of SEQ IDNOs: 1-68, or genetic region within, for example, 150 base pairs of oneof SEQ ID NO: 1-68 are amplified. In an embodiment, a single SNP withexceptionally high predictive value is amplified.

A pair of primers can be utilized in the amplification reaction. One orboth of the primers can be labeled, for example with a detectableradiolabel, fluorophore, or biotin molecule. The pair of primersincludes an upstream primer (which binds 5′ to the downstream primer)and a downstream primer (which binds 3′ to the upstream primer). Thepair of primers used in the amplification reactions are selectiveprimers which permit amplification of a size related marker locus.Primers can be selected to amplify a DNA including a SNP. Numerousprimers can be designed by those of skill in the art simply bydetermining the sequence of the desired target region, for example,using well known computer assisted algorithms that select primers withindesired parameters suitable for annealing and amplification.

If desired, an additional pair of primers can be included in theamplification reaction as an internal control. For example, theseprimers can be used to amplify a “housekeeping” nucleic acid molecule,and serve to provide confirmation of appropriate amplification. Inanother example, a target nucleic acid molecule including primerhybridization sites can be constructed and included in the amplificationreactor. One of skill in the art will readily be able to identify primerpairs to serve as internal control primers.

Primer Design Strategy:

Increased use of polymerase chain reaction (PCR) methods has stimulatedthe development of many programs to aid in the design or selection ofoligonucleotides used as primers for PCR. Four examples of such programsthat are freely available via the Internet are: PRIMER™ by Mark Daly andSteve Lincoln of the Whitehead Institute (UNIX, VMS, DOS, andMacintosh), Oligonucleotide Selection Program by Phil Green and LaDeanaHiller of Washington University in St. Louis (UNIX, VMS, DOS, andMacintosh), PGEN™ by Yoshi (DOS only), and Amplify by Bill Engels of theUniversity of Wisconsin (Macintosh only). Generally these programs helpin the design of PCR primers by searching for bits of knownrepeated-sequence elements and then optimizing the T_(m) by analyzingthe length and GC content of a putative primer. Commercial software isalso available and primer selection procedures are rapidly beingincluded in most general sequence analysis packages.

Designing oligonucleotides for use as either sequencing or PCR primersto detect requires selection of an appropriate sequence thatspecifically recognizes the target, and then testing the sequence toeliminate the possibility that the oligonucleotide will have a stablesecondary structure. Inverted repeats in the sequence can be identifiedusing a repeat-identification or RNA-folding programs. If a possiblestem structure is observed, the sequence of the primer can be shifted afew nucleotides in either direction to minimize the predicted secondarystructure. When the amplified sequence is intended for subsequencecloning, the sequence of the oligonucleotide can also be compared withthe sequences of both strands of the appropriate vector and insert DNA.A sequencing primer only has a single match to the target DNA. It isalso advisable to exclude primers that have only a single mismatch withan undesired target DNA sequence. For PCR primers used to amplifygenomic DNA, the primer sequence can be compared to the sequences in theGENBANK™ database to determine if any significant matches occur. If theoligonucleotide sequence is present in any known DNA sequence or, moreimportantly, in any known repetitive elements, the primer sequenceshould be changed.

Detection of Alleles:

The nucleic acids obtained from the sample can be genotyped to identifythe particular allele present for a marker locus. A sample of sufficientquantity to permit direct detection of marker alleles from the samplecan be obtained from the subject. Alternatively, a smaller sample isobtained from the subject and the nucleic acids are amplified prior todetection. Any target nucleic acid that is informative for a chromosomehaplotype can be detected. Generally, the target nucleic acidcorresponds to a SNP. Any method of detecting a nucleic acid moleculecan be used, such as hybridization and/or sequencing assays.

Hybridization is the binding of complementary strands of DNA, DNA/RNA,or RNA. Hybridization can occur when primers or probes bind to targetsequences such as target sequences within genomic DNA. Probes andprimers that are useful generally include nucleic acid sequences thathybridize (for example under high stringency conditions) with a nucleicacid sequence including the SNP of interest, but do not hybridize to areference allele, or that hybridize to the reference allele, but do nothybridize to the SNP. Physical methods of detecting hybridization orbinding of complementary strands of nucleic acid molecules, include butare not limited to, such methods as DNase I or chemical footprinting,gel shift and affinity cleavage assays, Southern and Northern blotting,dot blotting and light absorption detection procedures. The bindingbetween a nucleic acid primer or probe and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of thenucleic acid probe is melted from its target. A higher (T_(m)) means astronger or more stable complex relative to a complex with a lower (Tm).

Generally, complementary nucleic acids form a stable duplex or triplexwhen the strands bind, (hybridize), to each other by formingWatson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable bindingoccurs when an oligonucleotide molecule remains detectably bound to atarget nucleic acid sequence under the required conditions.

Complementarity is the degree to which bases in one nucleic acid strandbase pair with the bases in a second nucleic acid strand.Complementarity is conveniently described by percentage, that is, theproportion of nucleotides that form base pairs between two strands orwithin a specific region or domain of two strands. For example, if 10nucleotides of a 15-nucleotide oligonucleotide faun base pairs with atargeted region of a DNA molecule, that oligonucleotide is said to have66.67% complementarity to the region of DNA targeted.

In the present disclosure, “sufficient complementarity” means that asufficient number of base pairs exist between an oligonucleotidemolecule and a target nucleic acid sequence (such as a SNP) to achievedetectable and specific binding. When expressed or measured bypercentage of base pairs formed, the percentage complementarity thatfulfills this goal can range from as little as about 50% complementarityto full (100%) complementary. In general, sufficient complementarity isat least about 50%, for example at least about 75% complementarity, atleast about 90% complementarity, at least about 95% complementarity, atleast about 98% complementarity, or even at least about 100%complementarity. The qualitative and quantitative considerationsinvolved in establishing binding conditions that allow one skilled inthe art to design appropriate oligonucleotides for use under the desiredconditions is provided by Beltz et al. Methods Enzymol 100:266-285,1983, and by Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength (suchas the Na⁺ concentration) of the hybridization buffer will determine thestringency of hybridization. Calculations regarding hybridizationconditions for attaining particular degrees of stringency are discussedin Sambrook et al., (1989) Molecular Cloning: a laboratory manual,second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters9 and 11). The following is an exemplary set of hybridization conditionsand is not limiting:

Very High Stringency (Detects Sequences that Share at Least 90%Complementarity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share at Least 80%Complementarity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share at Least 50%Complementarity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Methods for labeling nucleic acid molecules so they can be detected arewell known. Examples of such labels include non-radiolabels andradiolabels. Non-radiolabels include, but are not limited to an enzyme,chemiluminescent compound, fluorescent compound (such as FITC, Cy3, andCy5), metal complex, hapten, enzyme, colorimetric agent, a dye, orcombinations thereof. Radiolabels include, but are not limited to, ¹²⁵I,³²P and ³⁵S. For example, radioactive and fluorescent labeling methods,as well as other methods known in the art, are suitable for use with thepresent disclosure. In one example, primers used to amplify thesubject's nucleic acids are labeled (such as with biotin, a radiolabel,or a fluorophore). In another example, amplified target nucleic acidsamples are end-labeled to form labeled amplified material. For example,amplified nucleic acid molecules can be labeled by including labelednucleotides in the amplification reactions.

Nucleic acid molecules corresponding to one or more SNPs or allelesincluding the SNP can also be detected by hybridization procedures usinga labeled nucleic acid probe, such as a probe that detects only onealternative allele at a marker locus. Most commonly, the target nucleicacid (or amplified target nucleic acid) is separated based on size orcharge and transferred to a solid support. The solid support (such asmembrane made of nylon or nitrocellulose) is contacted with a labelednucleic acid probe, which hybridizes to it complementary target undersuitable hybridization conditions to form a hybridization complex.

Hybridization conditions for a given combination of array and targetmaterial can be optimized routinely in an empirical manner close to theT_(m) of the expected duplexes, thereby maximizing the discriminatingpower of the method. For example, the hybridization conditions can beselected to permit discrimination between matched and mismatchedoligonucleotides. Hybridization conditions can be chosen to correspondto those known to be suitable in standard procedures for hybridizationto filters (and optionally for hybridization to arrays). In particular,temperature is controlled to substantially eliminate formation ofduplexes between sequences other than an exactly complementary allele ofthe selected marker. A variety of known hybridization solvents can beemployed, the choice being dependent on considerations known to one ofskill in the art (see U.S. Pat. No. 5,981,185).

Once the target nucleic acid molecules have been hybridized with thelabeled probes, the presence of the hybridization complex can beanalyzed, for example by detecting the complexes.

Methods for detecting hybridized nucleic acid complexes are well knownin the art. In one example, detection includes detecting one or morelabels present on the oligonucleotides, the target (e.g., amplified)sequences, or both. Detection can include treating the hybridizedcomplex with a buffer and/or a conjugating solution to effectconjugation or coupling of the hybridized complex with the detectionlabel, and treating the conjugated, hybridized complex with a detectionreagent. In one example, the conjugating solution includes streptavidinalkaline phosphatase, avidin alkaline phosphatase, or horseradishperoxidase. Specific, non-limiting examples of conjugating solutionsinclude streptavidin alkaline phosphatase, avidin alkaline phosphatase,or horseradish peroxidase. The conjugated, hybridized complex can betreated with a detection reagent. In one example, the detection reagentincludes enzyme-labeled fluorescence reagents or calorimetric reagents.In one specific non-limiting example, the detection reagent isenzyme-labeled fluorescence reagent (ELF) from Molecular Probes, Inc.(Eugene, Oreg.). The hybridized complex can then be placed on adetection device, such as an ultraviolet (UV) transilluminator(manufactured by UVP, Inc. of Upland, Calif.). The signal is developedand the increased signal intensity can be recorded with a recordingdevice, such as a charge coupled device (CCD) camera (manufactured byPhotometrics, Inc. of Tucson, Ariz.). In particular examples, thesesteps are not performed when radiolabels are used. In particularexamples, the method further includes quantification, for instance bydetermining the amount of hybridization.

Allele Specific PCR:

Allele-specific PCR differentiates between target regions differing inthe presence of absence of a variation or polymorphism. PCRamplification primers are chosen based upon their complementarity to thetarget sequence, such as nucleic acid sequence in a DNA including a SNP,a specified region of an allele including a SNP, or to the SNP itself.The primers bind only to certain alleles of the target sequence. Thismethod is described by Gibbs, Nucleic Acid Res. 17:12427 2448, 1989,herein incorporated by reference.

Allele Specific Oligonucleotide Screening Methods:

Further screening methods employ the allele-specific oligonucleotide(ASO) screening methods (e.g. see Saiki et al., Nature 324:163-166,1986). Oligonucleotides with one or more base pair mismatches aregenerated for any particular allele or haplotype block. ASO screeningmethods detect mismatches between one allele (or haplotype block) in thetarget genomic or PCR amplified DNA and the other allele (or haplotypeblock), showing decreased binding of the oligonucleotide relative to thesecond allele (i.e. the other allele) oligonucleotide. Oligonucleotideprobes can be designed that under low stringency will bind to bothpolymorphic forms of the allele, but which at high stringency, only bindto the allele to which they correspond. Alternatively, stringencyconditions can be devised in which an essentially binary response isobtained, i.e., an ASO corresponding to a variant form of the targetgene will hybridize to that allele (haplotype block), and not to thereference allele (haplotype block).

Ligase Mediated Allele Detection Method:

Ligase can also be used to detect point mutations, such as the tag SNPsdisclosed herein, in a ligation amplification reaction (e.g. asdescribed in Wu et al., Genomics 4:560-569, 1989). The ligationamplification reaction (LAR) utilizes amplification of specific DNAsequence using sequential rounds of template dependent ligation (e.g. asdescribed in Wu, supra, and Barany, Proc. Nat. Acad. Sci. 88:189-193,1990).

Denaturing Gradient Gel Electrophoresis:

Amplification products generated using the polymerase chain reaction canbe analyzed by the use of denaturing gradient gel electrophoresis.Different alleles (haplotype blocks) can be identified based on thedifferent sequence-dependent melting properties and electrophoreticmigration of DNA in solution. DNA molecules melt in segments, termedmelting domains, under conditions of increased temperature ordenaturation. Each melting domain melts cooperatively at a distinct,base-specific melting temperature (T_(M)). Melting domains are at least20 base pairs in length, and can be up to several hundred base pairs inlength.

Differentiation between alleles (haplotype blocks) based on sequencespecific melting domain differences can be assessed using polyacrylamidegel electrophoresis, as described in Chapter 7 of Erlich, ed., PCRTechnology, Principles and Applications for DNA Amplification, W. H.Freeman and Co., New York (1992).

Generally, a target region to be analyzed by denaturing gradient gelelectrophoresis is amplified using PCR primers flanking the targetregion. The amplified PCR product is applied to a polyacrylamide gelwith a linear denaturing gradient as described in Myers et al., Meth.Enzymol. 155:501-527, 1986, and Myers et al., in Genomic Analysis, APractical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95 139,1988. The electrophoresis system is maintained at a temperature slightlybelow the Tm of the melting domains of the target sequences.

In an alternative method of denaturing gradient gel electrophoresis, thetarget sequences can be initially attached to a stretch of GCnucleotides, termed a GC clamp, as described in Chapter 7 of Erlich,supra. In one example, at least 80% of the nucleotides in the GC clampare either guanine or cytosine. In another example, the GC clamp is atleast 30 bases long. This method is particularly suited to targetsequences with high T_(m)'s.

Generally, the target region is amplified by polymerase chain reaction.One of the oligonucleotide PCR primers carries at its 5′ end, the GCclamp region, at least 30 bases of the GC rich sequence, which isincorporated into the 5′ end of the target region during amplification.The resulting amplified target region is run on an electrophoresis gelunder denaturing gradient conditions. DNA fragments differing by asingle base change will migrate through the gel to different positions,which can be visualized by ethidium bromide staining.

Temperature Gradient Gel Electrophoresis:

Temperature gradient gel electrophoresis (TGGE) is based on the sameunderlying principles as denaturing gradient gel electrophoresis, exceptthe denaturing gradient is produced by differences in temperatureinstead of differences in the concentration of a chemical denaturant.Standard TGGE utilizes an electrophoresis apparatus with a temperaturegradient running along the electrophoresis path. As samples migratethrough a gel with a uniform concentration of a chemical denaturant,they encounter increasing temperatures. An alternative method of TGGE,temporal temperature gradient gel electrophoresis (TTGE or tTGGE) uses asteadily increasing temperature of the entire electrophoresis gel toachieve the same result. As the samples migrate through the gel thetemperature of the entire gel increases, leading the samples toencounter increasing temperature as they migrate through the gel.Preparation of samples, including PCR amplification with incorporationof a GC clamp, and visualization of products are the same as fordenaturing gradient gel electrophoresis. Single-Strand ConformationPolymorphism Analysis: Target sequences, such as alleles can bedifferentiated using single-strand conformation polymorphism analysis,which identifies base differences by alteration in electrophoreticmigration of single stranded PCR products, for example as described inOrita et al., Proc. Nat. Acad. Sci. 85:2766-2770, 1989. Amplified PCRproducts can be generated as described above, and heated or otherwisedenatured, to form single stranded amplification products.Single-stranded nucleic acids can refold or form secondary structureswhich are partially dependent on the base sequence. Thus,electrophoretic mobility of single-stranded amplification products candetect base-sequence difference between alleles.

Chemical or Enzymatic Cleavage of Mismatches:

Quantitative differences between target sequences, such as alleles, canalso be detected by differential chemical cleavage of mismatched basepairs, for example as described in Grompe et al., Am. J. Hum. Genet.48:212-222, 1991. In another method, differences between targetsequences, such as alleles, can be detected by enzymatic cleavage ofmismatched base pairs, as described in Nelson et al., Nature Genetics4:11-18, 1993. Briefly, genetic material from an animal and an affectedfamily member can be used to generate mismatch free heterohybrid DNAduplexes. As used herein, “heterohybrid” means a DNA duplex strandcomprising one strand of DNA from one animal, and a second DNA strandfrom another animal, usually an animal differing in the phenotype forthe trait of interest. Positive selection for heterohybrids free ofmismatches allows determination of small insertions, deletions or otherpolymorphisms.

Non-Gel Systems:

Other possible techniques include non-gel systems such as TaqMan™(Perkin Elmer). In this system oligonucleotide PCR primers are designedthat flank the mutation in question and allow PCR amplification of theregion. A third oligonucleotide probe is then designed to hybridize tothe region containing the base subject to change between differentalleles of the gene. This probe is labeled with fluorescent dyes at boththe 5′ and 3′ ends. These dyes are chosen such that while in thisproximity to each other the fluorescence of one of them is quenched bythe other and cannot be detected. Extension by Taq DNA polymerase fromthe PCR primer positioned 5′ on the template relative to the probe leadsto the cleavage of the dye attached to the 5′ end of the annealed probethrough the 5′ nuclease activity of the Taq DNA polymerase. This removesthe quenching effect allowing detection of the fluorescence from the dyeat the 3′ end of the probe. The discrimination between different DNAsequences arises through the fact that if the hybridization of the probeto the template molecule is not complete (there is a mismatch of someform) the cleavage of the dye does not take place. Thus only if thenucleotide sequence of the oligonucleotide probe is completelycomplimentary to the template molecule to which it is bound willquenching be removed. A reaction mix can contain two different probesequences each designed against different alleles that might be presentthus allowing the detection of both alleles in one reaction.

Non-PCR Based Allele Detection:

The identification of a DNA sequence can be made without anamplification step, based on polymorphisms including restrictionfragment length polymorphisms in a subject and a control, such as afamily member. Hybridization probes are generally oligonucleotides whichbind through complementary base pairing to all or part of a targetnucleic acid. Probes typically bind target sequences lacking completecomplementarity with the probe sequence depending on the stringency ofthe hybridization conditions. The probes can be labeled directly orindirectly, such that by assaying for the presence or absence of theprobe, one can detect the presence or absence of the target sequence.Direct labeling methods include radioisotope labeling, such as with ³²Por ³⁵S. Indirect labeling methods include fluorescent tags, biotincomplexes which can be bound to avidin or streptavidin, or peptide orprotein tags. Visual detection methods include photoluminescents, Texasred, rhodamine and its derivatives, red leuco dye and3,3′,5,5′-tetramethylbenzidine (TMB), fluorescein, and its derivatives,dansyl, umbelliferone and the like or with horse radish peroxidase,alkaline phosphatase and the like.

Hybridization probes include any nucleotide sequence capable ofhybridizing to a nucleic acid sequence wherein a polymorphism, such as atag SNP, and thus defining a genetic marker, including a restrictionfragment length polymorphism, a hypervariable region, repetitiveelement, or a variable number tandem repeat. Hybridization probes can beany gene or a suitable analog. Further suitable hybridization probesinclude exon fragments or portions of cDNAs or genes known to map to therelevant region of the chromosome.

Exemplary tandem repeat hybridization probes for use in the methodsdisclosed are those that recognize a small number of fragments at aspecific locus at high stringency hybridization conditions, or thatrecognize a larger number of fragments at that locus when the stringencyconditions are lowered.

The present methods can also be embodied in a device or a systemcomprising one or more such devices, which is capable of carrying outall or some of the method steps described herein.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Prenatal screening for Down's Syndrome and other chromosomalabnormalities is most effectively achieved using interventionalprocedures such as amniocentesis and chorionic villus sampling (CVS)that obtain fetal or placental cells (respectively) for karyotypeanalysis. Unfortunately, these procedures involve a risk of spontaneousabortion that has been reported to be as high as 1% (Mujezinovic andAlfirevic, Obst. Gyn. 110: 687-694, 2007) (although complication rateshave fallen recently) and are generally only routinely offered toexpectant mothers who have an elevated risk of carrying an aneuploidfetus. Minimally invasive screening protocols utilizing specific proteinmarkers detectable in maternal serum in combination with ultrasound havebeen in use for a number of years but these do not achieve desirablelevels of sensitivity and specificity and so are not definitive (Summerset al., J. Med. Screen. 10: 107-11, 2003a, Meier et al., Prenat. Diagn.23: 443-446, 2003, Summers et al., Obstet. Bynecol. 101: 1301-1308,2003b). Furthermore, these markers are only surrogates of the underlyinggenetic abnormality.

As described herein, a high-throughput approach was used to characterizeDNA methylation patterns of CVS and MBCs obtained during the firsttrimester of pregnancy. The biomarker discovery efforts on chromosomes13, 18 and 21, which are frequently found to be aneuploid in livebirths. A list of epigenetic biomarkers was established, wherein thesebiomarkers each display differential methylation patterns between thesetissues on human chromosomes 13, 18 and 21. A custom microarray is alsodisclosed. The data presented herein provide a useful and uniquecatalogue of tissue-specific methylation patterns that can be used forfetal diagnosis.

Human chromosomal abnormalities are relatively common, occurring inapproximately 10-30% of conceptions. It has been estimated that 1 in 300live-born infants are aneuploid and that this figure rises to 1 in 25for stillborn infants. By far the commonest form of aneuploidy istrisomy 21 resulting in Down syndrome, which occurs at an average rateof approximately 1 of 500 live births and 1 of 250 conceptions (Hook,Lancet 340: 1109, 1992, Crotty et al., May Clin. Proc. 67: 373-378,1992). Other common autosomal trisomies including trisomy 18 (Edwardsyndrome) and trisomy 13 (Patau syndrome) occur with birth incidences of11n 6,500 and 1 in 12,500 (Spencer, Am. J. Med. Med. Genet. C. Semin.Med. GEnet. 145C: 18-32, 2007, Hassold et al., Environ. Mol. Mutagen.28: 167-175, 1996).

Example 1 Materials and Methods

Tissue Handling and DNA Extraction:

All samples used in the studies described below were de-identifieddiscarded tissues. CVS samples were obtained between gestational weeks11 and 13. Samples were dissected under a microscope and separated fromany decidua or flecks of blood. The culture media was removed and thetissue placed in 1.5-2.0 mL microcentrifuge tubes before freezing at−80° C. until DNA was extracted. To extract the DNA, one 5 mm stainlesssteel bead and 180 μL buffer ATL (from Qiagen's DNEASY® Blood and Tissuekit) were added to each CVS sample. The samples were placed in theTISSUELYSER® (Qiagen) Adaptor set 2×24, and the TISSUELYSER® wasoperated for 20 seconds at 30 Hz. The DNA was then purified using theDNEASY® Blood and Tissue kit as per the manufacturer's protocol. MBCswere obtained between gestational weeks 11 and 13. DNA was extractedfrom the MBC's using a modified protocol previously described byIovannisci, et al., 2006 (Iovannisci et al., 2006), using reagents fromthe MASTUREPURE® DNA Purification Kit (Epincentre Technologies, Madison,Wis., Cat. No. MCD85201). Briefly, clotted blood (approximately 1 mL)was mixed with an equal volume (1 mL) of 2× Tissue and Cell LysisSolution, votexed for 10 s and combined with 2 mL Tissue and Cell LysisSolution (MASTUREPURE® kit) containing 25 ng/μL proteinase K. 2 mL ofMPC Protein Precipitation Reagent was added to the total volume (4 mL)of the lysed sample and vortex vigorously for 10-15 sec, after whichsamples were cooled on ice for 1 hour. Cell debris were then pelleted bycentrifugation (×2) for at least 30 min at 2000 g and supernatantstransferred to a new 50 mL conical tube. DNA was precipitated in 2volumes of isoproponal, purified by phenol/chloroform extraction andresuspended in 504 DNAse/RNAse free water.

Target DNA Preparation for Microarray Analysis:

Genomic DNA samples (3 μg) were digested for 2 hours at 37° C. with 50 UHpaII (New England Biolabs [NEB]) in 90 μL total reaction volume usingNEB buffer 4. A second aliquot of 50 U, 1 μL of buffer 4, and 4 μL waterwere added and digestion continued overnight (total reaction volume was100 μL). Mock digestion controls were included to monitor digestionefficiency. Following overnight digestion, reactions were digestedfurther with 5 uL (50 U) of TspRI (NEB) at 65° C. for three hours.Reactions were then incubated further with 75 U (0.75 μl) ExonucleaseIII (NEB) and incubated at 30° C. for 1 hour. Enzymatic activity wasthen nullified by heating at 70° C. for 20 min after which 50 U of RecJF(NEB) were added to remove single stranded DNA. Reactions were incubatedfor 30 min at 37° C. and inactivated at 65° C. for 20 min. Reactionswere then phenol-chloroform extracted and the DNA precipitated andresuspended in 21.2 μL nuclease-free de-ionized water. Finally,extracted genomic DNA was quantified and assessed for purity using aNanoDrop ND-1000 UV-VIS Spectrophotometer.

CGH Target Labeling and Hybridization:

Experimental and reference DNA were labeled with Cy3-dUTP and Cy5-dUTPrespectively, and vice versa for dye-swaps, using a BioPrime CGH GenomicLabeling kit per the manufacturer's protocol (Agilent). Hybridizationwas performed in a mix containing 50 μL of human Cot-1, 52 μL of Agilent10× blocking agent, 260 μL of Agilent 2× HiRPM hybridization buffer, and158 μL of the labeled DNA. The hybridization mix was heated to 95° C.for 3 minutes, then incubated at 37° C. for 30 minutes and applied ontothe active array area. Hybridization with gentle agitation was carriedout at 65° C. for 40 hours. After hybridization, the slides were washedin Oligo aCGH Wash Buffer 1 and Oligo aCGH Wash Buffer 2, followed byacetonitrile and Stabilization and Drying Solution (Agilent) per themanufacturer's protocol. The slides were scanned using an AgilentScanner and the data was analyzed using Agilent Feature Extractionsoftware 8.1 (Agilent). Visualization and comparison of the datasetswere done with CGH-Analytics 3.2 (Agilent).

Pyrosequencing:

500 ng of each MBC and CVS sample was bisulfite converted using the EZDNA® Methylation Kit (Zymo Research, Orange, Calif.) as permanufacturer's protocol. Each PCR reaction contained 1× AmpliTaq bufferGold, 2.0 mM MgCl₂, 0.5 mM dNTP, 0.2 μM each primer, 20 ng bisulfiteconverted DNA, and 1.5 U AmpliTaq Gold in a 504 reaction volume. The PCRprimers were purchased from Integrated DNA Technologies, Inc.(Coralville, Iowa), and the reverse primers biotinylated at their 5′end. PCR cycling conditions were: 95° for 10 min, 47×(95° for 15 s, Tmfor 30 s, 72° for 30 s), 72° for 7 min, 4° hold. Pyrosequencing wasperformed and analyzed on the above-amplified PCR products on a BiotagePSQ 96MA machine as per the manufacturer's protocol (Biotage Co.,Uppsala, Sweden). Unless otherwise noted, all reagents and consumableswere purchased directly from Biotage. In brief, 404 of a StreptavidinSepharose bead (GE Healthcare Co.)/binding buffer mixture were combinedwith 40 μL of each amplified PCR product in a V-well 96 well plate andmixed for ten minutes at maximum speed on a vortex. Each sequencingprimer was diluted in annealing buffer and this mixture placed in a wellin a PSQ 96 low plate. After mixing, the beads were captured using avacuum prep tool and then flushed with 70% ethanol, 0.2M NaOH, and washbuffer successively for 15 seconds each. Beads were released into thesequencing primer/annealing buffer solution by turning off the vacuumand placing the probes in this solution. The primers were annealed byheating to 95° for 2 minutes. Based on the sequence to be analyzed, thepyrosequencing cartridge was prepared, the plate loaded into themachine, and the pyrosequencing program initiated.

Sequenom Epityper Analysis:

PCR reactions were carried out in a 384 well format as follows. To eachreaction was added 1.42 μL ddH₂O, 0.5 μL 10× Hot Star Buffer (Qiagen)(15 mM MgCl₂, Tris-C1, KCl, (NH₄)₂SO4, pH 8.7), 0.04 μL dNTP mix (25 mMeach), 5 U/μL Hot Star Taq (Qiagen). Primers were then added to a finalconcentration (each) of 1 μM and 1 μL bisulphite converted DNA (1 ng/μLper reaction). Reactions were incubated as follows: 94° C. for 15minutes then 45 cycles of 94° C. for 20 seconds, 56° C. for 30 seconds(temperature adjusted according to primer Tm), 72° C. for 1 minutefollowed by 72° C. for 3 minutes. Reactions were then treated withShrimp alkaline phosphatase (SAP), in vitro transcribed and analyzedaccording to the manufacturer's instructions (Sequenom). Fullymethylated DNA controls were obtained from Millipore-(CpGenome™Universal Methylated DNA, part number S7821).

Statistical Methods:

Each custom Agilent array was hybridized with an HpaII digested sample(HpaII+) against the same sample without HpaII digestion (HpaII−). ThisHpaII+/− hybridization is designed to detect hypomethylated MspIrecognition sites. If the CpG dinucleotide in an MspI site recognitionsite is hypomethylated, the DNA segments containing this site will bedigested by HpaII; thus the signal from the HpaII− sample should bestronger than the signal from the corresponding HpaII+ sample, whichwill be selectively digested by HpaII. In other words, the log signalratio of HpaII− to HpaII+ should be positive. Based on this design, theMspI sites can be identified, where the CVS samples and the MBC sampleshave different methylation patterns in the following way:

First, MspI sites were identified that are hypomethylated in either theCVS samples or the MBC samples, but not in both. It can be tested if anMspI site is hypomethylated in a type of tissue by testing the logsignal ratios of the probe targeting that site. If the log signal ratioof HpaII− to HpaII+ is significantly above 0, the MspI site ishypomethylated. Next, MspI sites were identified that weredifferentially methylated between CVS and MBC. This is done by testingif the log ratio of HpaII+/HpaII− for a probe is the same in both CVSand MBC. If the log ratio is significantly different, the MspI site isdifferentially methylated. The intersection of the two sets of MspIsites identified in the two previous steps is the set of MspI sets withtissue specific methylation patterns.

The statistical tests used were based on the empirical Bayesian methoddescribed in Smyth (Smyth, Sat. Appl. Genet. Mol. Biol. 3: Article 3,2004), with false discovery rate (FDR) controlled at 5%. The data(green/red signal ratios) are normalized using the cyclic loss method(Bolstad et al., Bioinformatics 19: 185-193, 2003). All analyses wereperformed using the statistical computing package R.

Example 2 Detection of Methylated Sequences and Results

In order to generate a target library of DNA fragments that are enrichedfor methylated DNA sequences, an approach was used that takes advantageof the inability of HpaII, an isoschizomer of MspI, to cleave its CCGGrecognition sequence when the central CpG dinucleotide of that core siteis methylated (Shann et al., Gen. Res. 18: 791-801, 2008). Pre-digestionof the sample DNA with TspRI generates a 9 bp overhang that renders thesample resistant to digestion by exonuclease III. However, exonucleaseIII is able to digest HpaII-generated 2 bp overhangs and so fragmentsthat are unmethylated at HpaII (MspI) sites and therefore cleaved bythis enzyme are lost from the fragment library after digestion withexonuclease III. Sham-digestion of a control library without HpaIIprovides a comparative analysis between HpaII-digested and sham-digestedlibraries for the high-throughput determination of DNA methylationstatus. This procedure is summarized in FIG. 1.

A custom microarray was designed such that every MspI site onchromosomes 13, 18 and 21 is represented by two flanking 60 bpoligonucleotide probes. Each array contains 215,060 informative probes.Among them, 78,548 probes target 42,978 MspI/HpaII sites in chr18, with35,570 sites targeted by a matching pair of probes. Also, 46,675 probestarget 25,878 MspUHpaII sites in chr21, with 20,797 sites targeted by amatching pair of probes. Furthermore, 89,837 probes target 49285MspUHpaII sites in chr13, with 40,552 sites targeted by a matching pairof probes. The complete array design is provided in Supplementary FileF1 of Chu et al., “A microarray based approach for the identification ofepigentic biomarkers for the non-invasive diagnosis of fetal disease,”Prenatal Diagnosis 29: 1020-1030, 2009, published on-line Jul. 31, 2009;the manuscript and the on-line supplementary information is incorporatedby reference herein.

Each member of a probe pair matches one flanking sequence of an MspIrecognition site whilst the other member of the pair matches theopposite flanking sequence. Thus two non-overlapping probes are presentfor every MspI site. To minimize false positive and negative results apooling strategy was adopted for DNA samples prior to target librarypreparation in which two pools each of CVS and MBC were used, where eachpool contained samples from four individuals respectively. technicalreplicate experiments were also carried out using a dye-swappingapproach.

Using this approach, 6,311 MspI/HpaII sites were identified across allthree chromosomes that were differentially methylated between CVS andMBCs and reached statistical significance under the criteria describedin Materials and Methods. These included 1,272 sites on chromosome 21;2,297 on chromosome 18; and 2,742 on chromosome 13 respectively. Thesecan be considered to be tissue specific differentially methylated CpGsites (T-DMRs). Of these differentially methylated sites, 5,499 arehypomethylated in CVS versus MBC whereas only 812 are hypomethylated inMBC versus CVS. The entire list of T-DMRs is provided in Table S1 of Chuet al., “A microarray based approach for the identification of epigenticbiomarkers for the non-invasive diagnosis of fetal disease,” PrenatalDiagnosis 29: 1020-1030, 2009, published on-line Jul. 31, 2009; themanuscript and the on-line supplementary information is incorporated byreference herein.

Given that the diagnostic utility of methylation-specific quantitativeanalysis of fetal DNA requires that the target amplicon include apolymorphic marker for which the fetus is heterozygous (Tong et al.,2006), the data was analyzed for significant T-DMRs that are locatedwithin 150 bp of a known SNP. This is because the most attractivecandidate T-DMRs will be adjacent to SNPs that are highly polymorphicand therefore common amongst target patient populations (Tong et al.,2006). Of the CpG sites hypomethylated in CVS relative to MBC, 888, 755and 482 on chromosomes 13, 18 and 21 respectively, were found to bewithin 150 bp of a polymorphic SNP using a heterozygosity cut-off of0.25. Similarly, of the CpG sites hypomethylated in MBC relative to CVS,151, 115 and 107 were found to be within 150 bp of a polymorphic SNPusing a heterozygosity cut-off of 0.25. The top 15 T-DMRs within 150 bpof a polymorphic SNP are shown in Table 1 and the full list, ranked bystatistical significance, in Tables S2-1 through S2-6 of Chu et al., “Amicroarray based approach for the identification of epigentic biomarkersfor the non-invasive diagnosis of fetal disease,” Prenatal Diagnosis 29:1020-1030, 2009, published on-line Jul. 31, 2009; the manuscript and theon-line supplementary information is incorporated by reference herein.

TABLE 1 T-DMRs within 150 bp of a polymorphic SNP determined by customAgilent microarray analysis for chromosomes 21 (A and B), 18 (C and D)and 13 (E and F). Probe SNP rs.id Combined pval Unique ID Probe PositionA. Hypomethylated in MBC Versus CVS on Chromosome 21 11702354 2.01E−13202853 chr21: 34806349-34806290 11702354 2.01E−13 13099 chr21:34806348-34806407 11702450 3.30E−12 15805 chr21: 46528161-4652822011702450 3.30E−12 107388 chr21: 46528162-46528103 2839418 4.57E−11 88603chr21: 42192976-42193035 2839418 4.57E−11 172376 chr21:42192977-42192918 6517531 2.35E−10 172933 chr21: 39559604-395595456517531 2.35E−10 172764 chr21: 39559603-39559662 2824493 9.08E−10 130381chr21: 18087813-18087872 2824493 9.08E−10 115667 chr21:18087814-18087755 2835676 1.67E−09 195931 chr21: 37513226-375132852835676 1.67E−09 74314 chr21: 37513227-37513168 2823026 2.50E−09 170571chr21: 15346480-15346539 2823026 2.50E−09 125765 chr21:15346481-15346422 6517254 5.09E−09 120612 chr21: 35002142-350022016517254 5.09E−09 105469 chr21: 35002143-35002084 2070368 5.09E−09 105469chr21: 35002143-35002084 2070368 5.09E−09 120612 chr21:35002142-35002201 220269 5.55E−09 212124 chr21: 42357349-42357290 2202695.55E−09 165397 chr21: 42357348-42357407 B. Hypomethylated in CVS VersusMBC on Chromosome 21 225395 1.33E−14 31538 chr21: 42559967-42560026225395 1.33E−14 115974 chr21: 42559968-42559909 2837528 2.52E−14 136995chr21: 40533598-40533539 2837528 2.52E−14 1216 chr21: 40533597-405336562827557 1.33E−13 12999 chr21: 22839640-22839581 2827557 1.33E−13 178006chr21: 22839639-22839698 2822564 9.55E−13 188482 chr21:14608183-14608242 2822564 9.55E−13 73052 chr21: 14608184-14608125 2338952.13E−12 85116 chr21: 27247103-27247162 233895 2.13E−12 11559 chr21:27247104-27247045 9980448 3.89E−12 193547 chr21: 42712730-427127899980448 3.89E−12 58155 chr21: 42712731-42712672 2827384 3.07E−11 97870chr21: 22605322-22605263 2827384 3.07E−11 120832 chr21:22605321-22605380 2826052 6.62E−11 14852 chr21: 20457190-204572492826052 6.62E−11 2381 chr21: 20457191-20457132 9977149 1.02E−10 30513chr21: 15936203-15936144 9977149 1.02E−10 39772 chr21: 15936202-159362613453 1.16E−10 145449 chr21: 34741083-34741024 3453 1.16E−10 142382chr21: 34741082-34741141 C. Hypomethylated in MBC Versus CVS onChromosome 18 8083921 6.01E−12 90918 chr18: 58916958-58917017 80839216.01E−12 189198 chr18: 58916959-58916900 546680 5.81E−11 158354 chr18:30988045-30988104 546680 5.81E−11 76298 chr18: 30988046-30987987 48911591.53E−10 48439 chr18: 72230922-72230863 4891159 1.53E−10 89527 chr18:72230921-72230980 16978450 4.22E−10 211790 chr18: 41517172-415172313786395 4.22E−10 211790 chr18: 41517172-41517231 7245283 4.32E−10 174750chr18: 957430-957371 7245283 4.32E−10 169527 chr18: 957429-9574889945379 4.47E−10 57634 chr18: 10928196-10928255 9945379 4.47E−10 52550chr18: 10928197-10928138 12958513 1.06E−09 28434 chr18:41520963-41521022 16978452 1.06E−09 28434 chr18: 41520963-415210227228161 1.06E−09 28434 chr18: 41520963-41521022 16978452 1.06E−09 41831chr18: 41520964-41520905 12958513 1.06E−09 41831 chr18:41520964-41520905 7228161 1.06E−09 41831 chr18: 41520964-4152090511875350 1.57E−09 50589 chr18: 66307845-66307786 11152348 2.01E−09118255 chr18: 58349522-58349463 D. Hypomethylated in CVS Versus MBC onChromosome 18 12955286 2.23E−14 197827 chr18: 39459451-39459510 18525312.23E−14 197827 chr18: 39459451-39459510 1852531 2.23E−14 111977 chr18:39459452-39459393 12955286 2.23E−14 111977 chr18: 39459452-394593931244833 1.58E−13 206906 chr18: 67161475-67161534 1244833 1.58E−13 100157Chr18: 67161476-67161417 2923220 3.66E−13 98530 Chr18: 67061707-670616482923220 3.66E−13 114767 Chr18: 67061706-67061765 16977803 7.31E−13 15847Chr18: 39904531-39904590 16977803 7.31E−13 13551 Chr18:39904532-39904473 603884 7.62E−13 6351 Chr18: 50016366-50016425 6038847.62E−13 155051 Chr18: 50016367-50016308 4800573 8.57E−13 40020 Chr18:20313668-20313609 4800573 8.57E−13 165509 Chr18: 20313667-2031372612970409 9.62E−13 107335 Chr18: 23756169-23756228 12970409 9.62E−1397494 Chr18: 23756170-23756111 11663168 1.10E−12 173600 chr18:910151-910092 11663172 1.10E−12 173600 chr18: 910151-910092 116631721.10E−12 174485 chr18: 910150-910209 11663168 1.10E−12 174485 chr18:910150-910209 E. Hypomethylated in MBC Versus CVS on Chromosome 137317471 6.71E−13 10348 Chr13: 20518120-20518061 7317471 6.71E−13 44374Chr13: 20518119-20518178 3742160 5.09E−12 200018 chr13:105943751-105943810 3742160 5.09E−12 153614 chr13: 105943752-105943693206321 1.75E−11 94618 Chr13: 31888677-31888736 206321 1.75E−11 2447Chr13: 31888678-31888619 9579199 2.41E−11 70801 Chr13: 28062767-280627089578047 2.41E−11 70801 Chr13: 28062767-28062708 9578047 2.41E−11 202671Chr13: 28062766-28062825 9579199 2.41E−11 202671 Chr13:28062766-28062825 9506534 2.82E−11 173211 Chr13: 20145259-201453189506534 2.82E−11 72382 chr13: 20145260-20145201 1411551 8.55E−11 49844chr13: 109170896-109170837 1411551 8.55E−11 49787 chr13:109170895-109170954 4772302 1.50E−10 77302 chr13: 99968298-999682399515119 5.74E−10 73274 chr13: 109207221-109207162 9515119 5.74E−10197654 chr13: 109207220-109207279 9551454 1.09E−09 201286 chr13:27730895-27730954 9551454 1.09E−09 214291 chr13: 27730896-2773083717593586 2.72E−09 121914 chr13: 40694042-40693983 F. Hypomethylated inCVS Versus MBC on Chromosome 13 9542537 2.39E−13 16916 chr13:70392637-70392696 9542537 2.39E−13 193406 chr13: 70392638-703925797983181 3.47E−13 118841 chr13: 82628956-82628897 7983181 3.47E−13 113031chr13: 82628955-82629014 166710 5.70E−13 2591 chr13: 35186503-35186562166710 5.70E−13 103271 chr13: 35186504-35186445 9301803 6.28E−13 90142chr13: 91837064-91837005 9301804 6.28E−13 46449 chr13: 9837063-918371229301804 6.28E−13 90142 chr13: 91837064-91837005 9301803 6.28E−13 46449chr13: 91837063-91837122 2025675 1.06E−12 158650 chr13:70621925-70621984 2025675 1.06E−12 40595 chr13: 70621926-7062186711617606 2.44E−12 24847 chr13: 109322585-109322526 11617606 2.44E−12156269 chr13: 109322584-109322643 9535813 3.74E−12 62282 chr13:51453087-51453146 9316563 3.74E−12 18480 chr13: 51453088-514530299316563 3.74E−12 62282 chr13: 51453087-51453146 9535813 3.74E−12 18480chr13: 51453088-51453029 980094 4.48E−12 156466 chr13: 94960749-949608082389355 4.48E−12 19072 chr13: 94960750-94960691To confirm the accuracy of the microarray data, the differentialmethylation of a number of MspI sites was confirmed by pyrosequencing(FIG. 2A) and Mass Array/Epityper (FIG. 2B). Notably, a number of thesesites are flanked by additional CpGs that are also differentiallymethylated, as indicated in FIG. 2B. Furthermore, of 14 CpG islandspreviously suggested to be good candidates for development as biomarkersfor fetal aneuploidies on chromosome 21 (Chim et al., Clin. Chem. 54:500-511, 2008), 70% are found in genes that we identified in our data ascontaining T-DMRs (Table 2).

TABLE 2 Comparison between previously identified chromosome 21 T-DMRs(Chim et al., supra, 2008) and current data Gene Previously T-DMRIdentified by Identified as T-DMR Current Study (Chim et al., 2008)(probe unique ID) Methylated in CVS C21orf63 Yes (30351) andunmethylated in OLIG2 No MBC CBR1 No SIM2 Yes (72327) DSCAM Yes (88779)TRPM2 Yes (149601) C21orf29 Yes (19822) COL18A1 Yes (29860) Unmethylatedin CVS, RUNX1 Yes (32873) methylated in MBC PDE9A Yes (49044) HIST1H2BKNo HSF2BP Yes (98506) COL6A1 Yes (107986) COL6A2 Yes (135238)

In this study, the first trimester placental DNA methylome at itsmaternal interface was evaluated to identify potential biomarkers forthe minimally-invasive diagnosis of fetal genetic disease.Differentially methylated MspI recognition sequences (CCGG) on humanchromosome 13, 18 and 21 were identified using DNA extracted from CVScompared to DNA from maternal leukocyte samples. This was achieved usinga custom microarray designed for this purpose and novel computationaland statistical methods.

Thus, a comprehensive analysis of DNA methylation differences betweengenomic DNA isolated from first trimester human placental samples attheir maternal interface and gestational age-matched MBC samples wasperformed. These data provided candidate markers for further developmentin the context of fetal genetic diagnosis. These biomarkers are of use,as they exhibited differential methylation between CVS and MBCs and werefound with 150 bp of highly polymorphic SNPs.

It was first demonstrated in 1997 that Y chromosome DNA derived from amale fetus can be detected by PCR in maternal plasma and serum (Lo etal., Am. J. Hum. Genet. 62: 768-775, 1998b). This minimally invasiveapproach requires only a maternal blood sample. Fetal DNA constitutesapproximately 3-10% of total maternal plasma DNA and it has been shownthat this frequency is increased both in aneuploid and preeclampticpregnancies when compared to those that progress nominally (Lo et al.,Clin. Chem. 45: 1747-51, 1999a, Lo et al., Clin. Chem. 45: 184-188,1999b, Lun et al., Clin. Chem. 45: 1664-1672, 2008). Methods for thedetection of specific sex-linked fetal DNA mutations (Costa et al., N.Engl. J. Med. 346: 1502, 2002) and paternally inherited Beta-thalassemiaand achrondroplasia have been published (Li et al., JAMA 293: 843-849,2005, Li et al., Prenat. Diagnosis 27: 11-172007) and a plasma-based DNAtest to predict fetal Rhesus D blood group status is now widely used inclinical practice (Lo et al., NEJM 339: 1734-1738, 1998a).

Recently, differences in the methylation status of the RASSF1 and Maspingenes in the DNA of placental tissue and maternal hematopoetic cellshave been exploited via methylation-specific PCR to selectively amplifyfetal DNA sequences on chromosomes 3 and 18 respectively from maternalblood (Chim et al., PNAS 102: 14753-8, 2005, Chan et al., Clin. Chem.52: 2211-2218, 2006). This approach has been shown to have diagnosticpotential in the context of fetal disease in that elevated placentalRASSF1 levels in maternal plasma have been shown in early pregnancy tobe associated with an eventual diagnosis of preeclampsia (Tsui et al.,Prenat. Diagn. 27: 1212-1218, 2007b) and Maspin may have potential forthe detection of a subset of cases of trisomy 18 (Tong et al., Clin.Chem. 52: 2194-2202, 2006). One limitation to progression in this field,however, is the lack of comprehensive information relating to CpG sitesthat are differentially methylated between CVS and MBC (see Chim et al.,Clin Chem. 54: 500-11, 2008). Provided herein is a comprehensive list ofT-DMRs that exist between CVS and MBC on chromosomes with diagnosticsignificance in the context of fetal aneuploidy. Furthermore, withinthis context, sequences have been identified that detect aneuploidy in afetus. These sequences are provided herein.

Example 3 Detection of Fetal Aneuploidy

Recruitment of Pregnant Individuals During Early Gestation:

Patients who are >35 years of age or have a family history of birthdefects or genetic conditions are referred for genetic counseling as amatter of routine. During this consult they are generally offeredamniocentesis or CVS depending on gestational age and preference.Regardless of whether they elect to undergo an invasive diagnosticprocedure, the vast majority of these individuals receive a firsttrimester serum screen and an ultrasound for nuchal translucency.Patients who are <35 years of age and have no family history of birthdefects or genetic conditions are not routinely scheduled for geneticcounseling. Instead they undergo a first trimester serum screen and anuchal translucency test. If the patient has an increased risk result,she is contacted by a genetic counselor and is offered a consult anddiagnostic testing via amniocentesis or CVS. All study participants aredrawn from the population of individuals undergoing informed consent andpatient recruitment will take place in the Center for Medical Genetics.Should individuals who are consented elect not to undergo either CVS oramniocentesis, these deliveries are tracked through the GIS database(above) to obtain post-natal outcome data.

Data and Sample Tracking:

Each participant entering into the study is assigned a unique bar code,to be issued at the time of consent. This bar code is used to track thepatient's blood sample, plasma-derived DNA, sequencing library and theresulting data and is linked to all available clinical and demographicinformation including diagnostic test results and birth outcome.

Separation of Plasma from Whole Blood:

Whole blood is centrifuged at 1600×g for 13 min at 4° C., settingacceleration and deceleration to 3. 1 ml aliquots of plasma are pipettedinto 1.5 ml centrifuge tubes. Cellular debris is pelleted bycentrifuging the plasma at 16000×g for 10 min at 4° C. 900 ul from eachtube is pipetted into a clean 1.5 ml tube. Plasma aliquots are stored at−80° C.

DNA Extraction from Plasma:

DNA is extracted from plasma using the QIAMP® DNA mini kit (Qiagen) andsupplied reagents. Briefly, 1 vial of frozen plasma is thawed to roomtemperature and split into two tubes. 40 ul of Qiagen Protease is addedto each tube and the sample inverted 5 times to mix. 400 ul buffer AL isadded to each tube and the sample vortexed for 15 sec. Samples areincubated at 56° C. for 10 min. 400 ul 100% ethanol is added to eachtube and tubes are vortexed for 15 sec. 600 ul of mixture is applied tothe spin column and centrifuged at 6000×g for 1 min, repeating this stepas many times as necessary to get the entire plasma sample through thesame column. The column is washed by adding 500 ul buffer AW andcentrifuging for 1 min at 6000×g. The column is then washed again byadding 500 ul buffer AW2, and centrifuging for 4 min at maximum speed.To remove residual ethanol, the column is placed in a clean collectiontube and centrifuged at maximum speed for 2 min. To elute the DNA, thecolumn is placed in a 1.5 ml tube, 75 ul of RNase/DNase free water isadded to the column, the column is incubated at room temp for 5 minutesand then centrifuged for 1 min at 6000×g.

HpaII Digestion for Selection of Methylated DNA Loci:

DNA is combined with 4 ul 10× Fast Digestion Green buffer and 0.5 ulFast Digest HpaII (Fermentas, Glen Burnie, Md.) in a 40 ul reactionvolume and incubated at 37° C. for 5 min. An additional 0.5 ul FastDigest HpaII is added and the mixture is incubated for another 5 min at37 degrees. The reaction is then incubated at 70° C. for 5 minutes toheat inactivate the enzyme. For each sample, a mock digestion in whichno HpaII was added is run simultaneously. Samples are then purifiedusing Qiagen's MINELUTE® Reaction Clean-up Kit (Qiagen, Valencia,Calif.) as per the manufacturer's protocol. Each sample is combined with300 ul buffer ERC, applied to a spin column and centrifuged at maximumspeed for 1 min. The column is washed with 750 ul buffer PE and thentransferred to a 1.5 ml tube. DNA is eluted by adding 10 ul water to thecenter of the spin column, incubating for 1 minute at room temperatureand centrifuging at maximum speed for 1 min), see FIG. 3.

Bisulphite Treatment of DNA:

DNA can be bisulfite converted using the EZ DNA® Methylation Kit (ZymoResearch, Irvine, Calif.) as per manufacturer's protocol. Briefly, 500ng DNA is combined with 5 ul M-Dilution buffer in a total reactionvolume of 50 ul and incubated at 37 degrees for 15 min. 100 ul of the CTConversion Reagent is added and the mixture incubated at 50 degrees for16 hr. After incubating on ice for 10 min, the sample is combined with400 ul M-Binding Buffer in a spin column. The column is inverted severaltimes to mix and then centrifuged at maximum speed for 30 sec. Afterwashing the column with 100 ul M-Wash Buffer, 200 ul M-desulphonationbuffer is added to the column and incubated at room temperature for 20min. The column is centrifuged at maximum speed for 30 sec and thenwashed twice with 200 ul M-Wash Buffer. After transferring the column toa 1.5 ml tube, the DNA is eluted by adding 10 ul M-Elution Buffer andcentrifuging at maximum speed for 30 sec.

MBD2 Protein Mediated Enrichment of Methylated DNA:

Enrichment of DNA by methylation status is performed using theMETHYLMINER® Methylated DNA Enrichment Kit (Invitrogen, Carlsbad,Calif.) as per manufacturer's protocol. Briefly, 10 ul of DYNABEADS®M-280 Streptavidin beads is washed with 90 ul binding/wash buffer andthen resuspended in 100 ul binding/wash buffer. 3.5 ug of MBD-BiotinProtein in 100 ul binding/wash buffer is combined with the beads, andthe mixture is incubated at room temperature for 1 hr on a rotatingmixer. The beads are washed three times in wash/binding buffer and thenresuspended in 100 ul binding/wash buffer. Up to 1 ug fragmented DNA in100 ul binding/wash buffer is combined with the beads, and this mixtureis incubated on a rotating mixer for 1 hour at room temperature. Thebeads are collected against the side of the tube by a magnet and thesupernatants removed. This fraction contains the non-methylated DNA. Thebeads are washed twice with 200 ul bind/wash buffer. The beads then areincubated in 200 ul 2M NaCl on a rotating mixer for 3 min at roomtemperature, collected against the side of the tube by a magnet and thesupernatant containing the methylated DNA pipetted into a clean tube.The beads are incubated with an additional 200 ul 2M NaCl to ensuretotal collection of the methylated DNA. DNA is then purified andconcentrated by ethanol precipitation and resuspended in water.

Enzymatic Enrichment of Methylated DNA Using TspRI and Exonuclease:

This method can be used prior to a gene/locus specific amplification orother enrichment step. This method selectively isolates methylated DNAfragments from a complex DNA sample. It is highly effective in thisregard and can be used prior to simple amplification/enrichment andsubsequent copy number analysis. Genomic DNA samples (3 μg) are digestedfor 2 hours at 37° C. with 50 U HpaII (New England Biolabs) in 904 totalreaction volume using NEB buffer 4. A second aliquot of 50 U, 1 μL ofbuffer 4, and 44 water is added and digestion continued overnight (totalreaction volume was 1004). Mock digestion controls are included tomonitor digestion efficiency. Following overnight digestion, reactionsare digested further with 5 uL (50 U) of TspRI (NEB) at 65° C. for threehours. Reactions are then incubated further with 75 U (0.75 μl)Exonuclease III (NEB) and incubated at 30° C. for 1 hour. Enzymaticactivity is then nullified by heating at 70° C. for 20 min after which50 U of RecJF (NEB) are added to remove single stranded DNA. Reactionsare incubated for 30 min at 37° C. and inactivated at 65° C. for 20 min.Reactions are then phenol-chloroform extracted and the DNA precipitatedand resuspended in 21.24 nuclease-free de-ionized water. Finally,extracted genomic DNA is quantified and assessed for purity using aNanoDrop ND-1000 UV-VIS Spectrophotometer.

Illumina DNA Sequencing Sample/DNA Library Preparation:

Plasma is separated from whole blood following centrifugation at 1,600×gfor 10 minutes, followed by a second centrifugation to removecontaminating nucleated cells at 16,000×g for 10 minutes. DNA isextracted from plasma using the QIAAMP® DNA Blood Mini kit (Qiagen) andan Illumina sequencing library prepared as follows. The following oligos5′—ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC*T—3′ (SEQ ID NO: 69) and5′-/5Phos/GAT CGG AAG AGC TCG TAT GCC GTC TTC TGC TTG—3′ (SEQ ID NO: 70)are resuspended in TE and annealed in 1×T4 DNA Ligase Reaction Buffer(NEW ENGLAND BIOLABS®, NEB) by heating at 95° C. for 5 minutes and thenslowly cooled to room temperature for a final concentration of 36 mMannealed adaptor. Plasma DNA fragments is end repaired and then terminalA-residues added using the NEBNext End Repair and the NEBNext dA-tailingmodules as per manufacturer's protocols (NEB). Following reactioncleanup using the MINELUTE® Cleanup kit (Qiagen), DNA fragments iscombined with 0.5 uM adaptor and 400 U T4 DNA ligase (NEB) and incubatedfor 2 hours at 16° C. After reaction cleanup with MINELUTE® Cleanup kit,PCR is performed using the following primers: 5′—CAA GCA GAA GAC GGC ATACGA GCT CTT CCG ATC*T—3′ (SEQ ID NO: 71) and 5′—AAT GAT ACG GCG ACC ACCGAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC*T—3′ (SEQ ID NO: 72)and Phusion High-Fidelity DNA Polymerase (NEB). PCR conditions willinclude an initial denaturation (98° C. 30 s), 12 cycles of 98° C. for10 s, 65° C. for 30 s and 72° C. for 30 s, with a final extension of 72°C. for 7 min. Following amplification, the PCR reaction is cleaned upusing the MINELUTE® PCR Purification Kit (Qiagen).

Illumina DNA Sequencing Sample/DNA Library Preparation:

Plasma is separated from whole blood following centrifugation at 1,600×gfor 10 minutes, followed by a second centrifugation to removecontaminating nucleated cells at 16,000×g for 10 minutes. DNA isextracted from plasma using the QIAAMP® DNA Blood Mini kit (Qiagen) andan Illumina sequencing library prepared as follows. The following oligos5′—ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC*T—3′ (SEQ ID NO: 73) and5′-/5Phos/GAT CGG AAG AGC TCG TAT GCC GTC TTC TGC TTG—3′ (SEQ ID NO: 74)are resuspended in TE and annealed in 1×T4 DNA Ligase Reaction Buffer(NEB) by heating at 95° C. for 5 minutes and then slowly cooled to roomtemperature for a final concentration of 36 mM annealed adaptor. PlasmaDNA fragments is end repaired and then terminal A-residues added usingthe NEB NEXT END REPAIR® and the NEBNEXT® dA-tailing modules as permanufacturer's protocols (NEB). Following reaction cleanup using theMINELUTE® Cleanup kit (Qiagen), DNA fragments are combined with 0.5 uMadaptor and 400 U T4 DNA ligase (NEB) and incubated for 2 hours at 16°C. After reaction cleanup with MINELUTE® Cleanup kit, PCR is performedusing the following primers: 5′—CAA GCA GAA GAC GGC ATA CGA GCT CTT CCGATC*T—3′ (SEQ ID NO: 75) and 5′—AAT GAT ACG GCG ACC ACC GAG ATC TAC ACTCTT TCC CTA CAC GAC GCT CTT CCG ATC*T—3′ (SEQ ID NO: 76) and PHUSION®High-Fidelity DNA Polymerase (NEB). PCR conditions include an initialdenaturation (98° C. 30 s), 12 cycles of 98° C. for 10 s, 65° C. for 30s and 72° C. for 30 s, with a final extension of 72° C. for 7 min.Following amplification, the PCR reaction is cleaned up using theMINELUTE® PCR Purification Kit (Qiagen).

Pyrosequencing:

Specific details of Pyrosequencing analysis vary depending on thecontext of the desired analysis. Examples \ of pyrosequencing basedmethods that are appropriate for digital analysis of locus-specific DNAcopy number are provided below.

Library Preparation:

Prior to the following steps, a variety of methods can be utilized togenerate targeted fetal DNA fragments on chromosomes of interest.Fragments are then processed as follows. Fragment ends are polished byincubation in polishing reaction (1× Polishing Buffer, dNTP's, ATP,polynucleotide kinase and T4 DNA polymerase) at 12° C. for 15 minutesand 25° C. for 15 minutes. Polished fragments are cleaned via a secondMINELUTE® PCR purification column. Adapters ‘A’ and ‘B’ are ligated tothe polished DNA fragments by incubation at 25° C. for 15 minutes.Ligation reaction is purified via MINELUTE® PCR purification column. DNAfragments with 2 ‘A’ or 2 ‘B’ adapters are removed using streptavidincoated library immobilization beads. ‘B’ adapters are biotinylated andthus adhere to the coated beads, while ‘A’ adapters do not. Fragmentswith ‘A’ adapters on both ends are washed away with BEAD WASH® buffer.Following bead washing DNA is denatured and the single unbound strand of‘A’ adapter and ‘B’ complement is retained for future processing.Strands with ‘B’ adapters on both ends do not have an unbound strand andso are left on the beads at this point. A 1 ml aliquot of final singlestranded product is run on a bioanalyzer RNA 6000 pico chip to assesssize distribution and concentration.

emPCR:

sstDNA fragments are bound to library capture beads by mixing samplelibrary with capture beads and performing the following thermocyclerprogram: 80° C. for 5 minutes, ramp 0.1° C./sec to 70° C., hold at 70°C. for 1 minute, ramp 0.1° C./sec to 60° C., hold at 60° C. for 1minutes, ramp 0.1° C./sec to 50° C. hold at 50° C. for 1 minute, ramp0.1° C./sec to 20° C. Emulsion of PCR reagents in microreactors withlibrary capture beads is prepared by mixing beads, PCR reaction mix (1×amplification mix, amplification primers, 0.15 U/ml PLATINUM TAQ®(Invitrogen)), and emulsion oil and mixing vigorously using a TISSUELYSER® (Qiagen). Emulsion is distributed into a PCR plate and templateamplification is carried out in a thermocycler using the followingcycling conditions: Hotstart activation for 4 minutes at 94° C., 40cycles of 94° C. for 30 seconds, 58° C. for 1 minute, 68° C. for 90seconds followed by 13 cycles of 94° C. for 30 seconds and 58° C. for 6minutes. Following template amplification, emulsions are broken andbeads with amplified product recovered by repeated washes with ethanolusing a syringe filter unit.

Bead Enrichment:

Enrichment beads are added to the recovered amplification beads.Enrichment beads are coated with oligos complementary to the free end ofthe amplified template. Successful amplification beads become bound tothe paramagnetic enrichment beads and are drawn out of solution using amagnetic rack. Unsuccessful amplification beads are drawn off with thesupernatant and discaraded. The bond between the amplification andenrichment beads is broken using 125 mM NaOH. Enrichment beads arepelleted using a magnetic rack and the enriched amplification beads arerecovered. Melt solution is neutralized by repeated washes with 1×Annealing buffer and the beads left suspended in annealing buffer.

Sequencing primers are added to the mixture of beads and annealingbuffer and annealed to the template using the following thermocyclerconditions: 65° C. for 5 minutes, ramp to 50° C. at 0.1° C./second, holdat 50° C. for 1 minute, ramp to 40° C. at 0.1° C./second, hold at 40° C.for 1 minute, ramp to 15° C. at 0.1° C./second, hold 15° C. Beads arecounted using a Beckman Z1 particle counter.

PICOTITERPLATE® Preparation:

Based on bead count obtained above and manufacturer recommendations forthe PICOTITERPLATE® region size being used, control beads and samplebeads are mixed to form the sequencing sample. Packing beads, samplebeads and enzyme beads are applied to the PICOTITERPLATE® as permanufacturer instructions.

Sequencing Reaction:

The PICOTITERPLATE® is loaded onto the FLX® sequencer and the runstarted. The FLX® uses pyrosequencing chemistry and detects theincorporation of each nucleotide in real time.

emPCR Titration:

For each sample, a preliminary titration run must be performed in orderto determine the best ratio of DNA template to amplification beads toobtain a maximum amount of usable sequence from the final data run. Theconcentration of DNA template is determined in copies/ml by applying themass of the average fragment size as determined from the bioanalyzeroutput to the measured concentration of sstDNA library. emPCR reactionscorresponding to titration points of 0.5, 2, 4 and 16 copies/bead areperformed as described for emPCR above. Emulsions are broken and beadsrecovered. Amplification beads are not enriched but are counted andloaded onto a pico titer plate for sequencing analysis. The number ofbeads that can be successfully sequenced is used to determine theoptimum ratio of DNA to beads for the final emPCR reaction to producetemplate for data production.

DNA Copy Number Analysis by Real Time PCR:

TAQMAN® Copy Number assays for each target gene as well as the TAQMAN®Copy Number Reference assay may be purchased from Applied Biosystems.For each real time PCR reaction, 10 ul 2× TAQMAN® Genotyping Master Mix,1 ul 20× TAQMAN® Copy Number assay, 1 ul 20× TAQMAN® Copy NumberReference assay, 20 ng DNA and water are combined for a total reactionvolume of 20 ul. Each sample is run in triplicate for quality control.Cycling conditions were 95 degrees for 10 min followed by 40 cycles of95 degrees for 15 sec and 60 degrees for 1 min. The real time PCRreactions are read and analyzed using COPYCALLER® Software on the 7900HTSequence Detection System (Applied Biosystems), see FIG. 3.

Tissue Handling and DNA Extraction:

DNA can be recovered from placental tissues or blood cells to confirmDNA methylation levels in pure sample. Placental samples are dissectedunder a microscope and separated from any decidua or flecks of blood.The culture media is removed and the tissue placed in 1.5-2.0 mLmicrocentrifuge tubes before freezing at −80° C. until DNA is extracted.To extract the DNA, one 5 mm stainless steel bead and 1804 buffer ATL(from Qiagen's DNEASY® Blood and Tissue kit) were added to each CVSsample. The samples are placed in the TISSUELYSER® (Qiagen) Adaptor set2×24, and the TISSUELYSER® operated for 20 seconds at 30 Hz. The DNA isthen purified using the DNEASY® Blood and Tissue kit as per themanufacturer's protocol. MBCs are obtained between gestational weeks 11and 13. DNA is extracted from the MBC's using a modified protocolpreviously described by Iovannisci, et al., 2006 (25), using reagentsfrom the MASTUREPURE® DNA Purification Kit (Epincentre Technologies,Madison, Wis., Cat. No. MCD85201). Briefly, clotted blood (approximately1 mL) is mixed with an equal volume (imp of 2× Tissue and Cell LysisSolution, votexed for 10 s and combined with 2 mL Tissue and Cell LysisSolution (MASTUREPURE® kit) containing 25 ng/μL proteinase K. 2 mL ofMPC Protein Precipitation Reagent is added to the total volume (4 mL) ofthe lysed sample and vortex vigorously for 10-15 sec, after whichsamples are cooled on ice for 1 hour. Cell debris are then pelleted bycentrifugation (×2) for at least 30 min at 2000 g and supernatantstransferred to a new 50 mL conical tube. DNA is precipitated in 2volumes of isoproponal, purified by phenol/chloroform extraction andresuspended in 50 μL DNAse/RNAse free water.

Quantification of Fetal DNA Frequency Using Real Time PCR:

To validate fetal DNA frequency in maternal plasma of samples in aclinical trial, Real Time PCR determination of fetal DNA concentrationin maternal plasma is carried according to the method of Maron, et al(Maron, 2007) using the following primers:

SRY: Forward primer {SEQ ID NO: 77) 5′-TCCTCAAAAGAAACCGTGCAT-3′Reverse primer (SEQ ID NO: 78) 5′-AGATTAATGGTTGCTAAGGACTGGAT-3′ Probe-(SEQ ID NO: 79) 5′-FAM-CACCAGCAGTAACTCCCCACAACCTCTTT-TAMRA-3′B-globin: Forward primer (SEQ ID NO: 80) 5′-GTGCACCTGACTCCTGAGGAGA-3′Reverse primer- (SEQ ID NO: 81) 5′-CCTTGATACCAACCTGCCCAG-3′ Probe-(SEQ ID NO: 82) 5′-FAM-AAGGTGAACGTGGATGAAGTTGGTGG-TAMRA-3′

B-globin is an ubiquitous housekeeping gene and is run concurrently withthe SRY to ensure that DNA was present for each sample, irrespective offetal gender. In order to estimate DNA concentration in the plasma DNA,a standard curve is run simultaneously alongside the plasma DNA sample.The standard curve DNA is prepared using commercially available DNA ofknown concentration. The range of values for the standard curve isapproximately 6.4 μg/5 ul to 20,000 μg/5 ul. For each real time PCRreaction, 12.5 ul 2× TAQMAN® Universal PCR Master Mix, 1.25 ul 10 uMforward primer, 1.25 ul 10 uM reverse primer and 0.0625 ul 100 uM probe,10 ul plasma DNA, 5 ul standards or 10 ul water (to serve as negativecontrol) is added to the appropriate wells. Each plasma DNA sample andthe negative control are run in triplicate. The standard curve DNA willalso be run in triplicate. The thermal cycling conditions involve aninitial denaturation step of 95° C. for 10 minutes, followed by 50cycles of 95° C. for 15 sec and 60° C. for 1 min. The real time PCRreactions are performed using the 7900HT Sequence Detection System(Applied Biosystems).

Sequenom Epityper Analysis:

DNA methylation can be abalyzed in a quantitative fashion using the MassArray method from SEQUENOM®. This is not suitable for digital chromosomecopy number analysis but rather serves as a suitable method forconfirming DNA methylation levels in samples of DNA. This method wasused to validate results obtained by microarray based discovery (Chu etal., 2009, supra) of differentially methylated fetal versus maternalDNA. PCR reactions are carried out in a 384 well format as follows. Toeach reaction is added 1.42 μL ddH₂O, 0.54 μL 10× HOT STAR® Buffer(Qiagen) (15 mM MgCl₂, Tris-Cl, KCl, (NH₄)₂SO4, pH 8.7), 0.044 dNTP mix(25 mM each), 5 U/μL HOT STAR® Taq (Qiagen). Primers are then added to afinal concentration (each) of 1 μM and 14 bisulphite converted DNA (1ng/4 per reaction). Reactions are incubated as follows: 94° C. for 15minutes then 45 cycles of 94° C. for 20 seconds, 56° C. for 30 seconds(temperature adjusted according to primer Tm), 72° C. for 1 minutefollowed by 72° C. for 3 minutes. Reactions are then treated with Shrimpalkaline phosphatase (SAP), in vitro transcribed and analyzed accordingto the manufacturer's instructions (Sequenom). Fully methylated DNAcontrols are obtained from Millipore-(CpGenome Universal Methylated DNA,part number S7821).

It is apparent that the precise details of the methods or compositionsdescribed may be varied or modified without departing from the spirit ofthe described invention. We claim all such modifications and variationsthat fall within the scope and spirit of the claims below.

The invention claimed is:
 1. A method of detecting aneuploidy ofchromosome 21 in human fetal genomic DNA from a human fetus, comprising:(a) obtaining purified human fetal genomic DNA from a human maternalbiological sample comprising fetal genomic DNA and maternal DNA,comprising separating a fetal CpG-containing genomic sequence fromchromosome 21 comprising at least 15 consecutive nucleotides of SEQ IDNO: 1 comprising the CpG at position 153-154 from said maternal DNA,wherein the CpG at position 153-154 is not methylated on said maternalDNA and is methylated on said fetal DNA; and (b) detecting an aneuploidcopy number of the CpG-containing genomic sequence from chromosome 21 inthe purified human fetal genomic DNA obtained in step (a).
 2. The methodof claim 1, wherein detecting the aneuploid copy number of theCpG-containing genomic sequence comprises detecting an allelic ratio ofa bi-allelic single nucleotide polymorphism in the purified human fetalgenomic DNA, wherein an allelic ratio of 1:2 or 2:1 is detected.
 3. Themethod of claim 2, wherein SEQ ID NO: 1 comprises the single nucleotidepolymorphism.
 4. The method of claim 1, wherein detecting the aneuploidcopy number of the CpG-containing genomic sequence comprises detectingan allelic ratio of a biallelic short tandem repeat polymorphism in thepurified human fetal genomic DNA, wherein an allelic ratio of other than1:1 is detected.
 5. The method of claim 1, wherein obtaining purifiedhuman fetal genomic DNA from a human maternal biological samplecomprises the use of a microarray.
 6. The method of claim 1, whereinobtaining purified human fetal genomic DNA from a human maternalbiological sample comprises the use of a restriction enzyme thatdifferentially cleaves methylated or unmethylated DNA.
 7. The method ofclaim 6, wherein the restriction enzyme is Hpa II.
 8. The method ofclaim 1, wherein obtaining purified human fetal genomic DNA from a humanmaternal biological sample comprises the use of bisulfite.
 9. The methodof claim 1, further comprising amplifying the purified human fetalgenomic DNA.
 10. The method of claim 1, wherein obtaining purified humanfetal genomic DNA from a human maternal biological sample comprisesseparating a fetal CpG-containing genomic sequence from chromosome 21comprising SEQ ID NO:
 1. 11. The method of claim 1, wherein detecting ananeuploid copy number comprises at least one of 1) DNA amplification; 2)detecting a fluorescent signal; 3) detecting hybridization of a probe;and 4) DNA sequencing.
 12. The method of claim 1, wherein the aneuploidyis trisomy
 21. 13. The method of claim 1, wherein the human maternalbiological sample is a maternal blood sample.
 14. The method of claim 1,wherein the human fetus is between 11 and 13 weeks of age.
 15. Themethod of claim 1, further comprising karyotyping the human fetus.